Compositions and methods for promoting or inhibiting angiogenesis

ABSTRACT

Compounds, compositions and methods for promoting or inhibiting angiogenesis, and screening methods for identifying compounds are disclosed. The compounds bind to F1 ATP synthase particularly to the alpha and/or beta subunits of F1 ATP synthase. When bound to these subunits, they can function as angiostatin agonists, antagonists, partial agonists, inverse agonists, or allosteric modulators. When the compounds mimic or enhance the activity of angiostatin, they inhibit angiogenesis. When the compounds inhibit the ability of angiostatin to bind F1 ATP synthase and are either inactive at inhibiting angiogenesis or directly promote angiogenesis, or if they inhibit the activity of angiostatin, they promote angiogenesis. The compounds can be, for example, antibodies, antibody fragments, enzymes, peptides, nucleic acids such as oligonucleotides, or small molecules. The antibodies can be monoclonal, humanized, or polyclonal antibodies. The compounds can be conjugated to or combined with various cytotoxic agents and/or labeled compounds. Methods for promoting angiogenesis can be used to introduce vasculature to areas in a patient that can benefit from such increased vasculature. Methods for inhibiting angiogenesis can be used to treat disorders mediated by angiogenesis, for example, tumors, autoimmune disorders such as rheumatoid arthritis, and the like.

PRIORITY

This application is a continuation-in-part of U.S. Ser. No. 09/314,159,filed May 19, 1999, entitled “Angiostatin Receptor.” U.S. Ser. No.09/314,159, which claims priority to U.S. Ser. No. 60/086,155, filed onMay 19, 1998 and U.S. Ser. No. 60/124,070, filed on March 12, 1999. Thecontents of each of these applications is hereby incorporated byreference for all purposes.

FIELD OF THE INVENTION

This application is generally in the area of compositions and methodsfor promoting or inhibiting angiogenesis.

BACKGROUND OF THE INVENTION

Angiogenesis is the formation of new capillary blood vessels leading toneovascularization. Angiogenesis is a complex process which includes aseries of sequential steps including endothelial cell-mediateddegradation of vascular basement membrane and interstitial matrices,migration of endothelial cells, proliferation of endothelial cells, andformation of capillary loops by endothelial cells.

Both controlled and uncontrolled angiogenesis are thought to proceed ina similar manner. Endothelial cells and pericytes, surrounded by abasement membrane, form capillary blood vessels. Angiogenesis beginswith the erosion of the basement membrane by enzymes released byendothelial cells and leukocytes. The endothelial cells, which line thelumen of blood vessels, then protrude through the basement membrane.

Angiogenic stimulants induce the endothelial cells to migrate throughthe eroded basement membrane. The migrating cells form a “sprout” offthe parent blood vessel, where the endothelial cells undergo mitosis andproliferate. The endothelial sprouts merge with each other to formcapillary loops, thereby creating the new blood vessel.

In normal physiological processes such as wound healing, angiogenesis isturned off once the process is completed. In contrast, tumorangiogenesis is not self-limiting. The progressive growth of solidtumors beyond clinically occult sizes (e.g., a few mm³) requires thecontinuous formation of new capillary blood vessels to deliver nutrientsand oxygen for the tumor itself to grow, a process known as tumorangiogenesis. Solid tumors elicit an angiogenic response in thesurrounding normal tissue for further growth. The resultantneovascularization of the tumor is associated with more rapid growth,and local invasion. Therefore, either inhibition of tumor angiogenesis(antiangiogenic therapy) or selective destruction of a tumor's existingblood vessels (vascular targeting therapy) would suppress or arresttumor growth and its spread.

Further, in certain pathological (and nonmalignant) processes,angiogenesis is abnormally prolonged. Examples include ocularneovascular disease, which is characterized by invasion of new bloodvessels into the retina or cornea, as well as other eye-relateddiseases. Other angiogenesis-associated diseases include diabeticretinopathy and chronic inflammatory diseases such as rheumatoidarthritis, dermatitis and atherosclerosis.

Antiangiogenic therapy has been proposed for modulating suchangiogenesis-associated disorders. One approach has been to administerVEGF (vascular endothelial growth factor) inhibitors. Other approachesinvolve using angiostatin or endostatin, which are both known to inhibitangiogenesis. The in vivo use of angiostatin or endostatin is somewhatlimited by their relatively short half-lives in vivo.

It would be advantageous to have new antiangiogenic compositions andmethods to add to the arsenal of therapies available for treating theseangiogenesis-mediated disorders. It would also be advantageous to havenew methods for identifying such compositions and methods. The presentinvention provides such compositions and methods.

SUMMARY OF THE INVENTION

The present invention is directed to compounds, compositions and methodsfor promoting or inhibiting angiogenesis, and results from the discoverythat angiostatin binds to ATP synthase, in particular, to the alpha andbeta subunits of F1 ATP synthase, and, when so bound, inhibitsangiogenesis.

The compounds bind to F1 ATP synthase, particularly to the alpha and/orbeta subunits of F1 ATP synthase. When bound to these subunits, theyinhibit the ability of angiostatin to bind to the subunits, and canfunction as angiostatin agonists, antagonists, partial agonists, inverseagonists, or allosteric modulators. Compounds that mimic or enhance theactivity of angiostatin inhibit angiogenesis. Compounds that areinactive at inhibiting angiogenesis, directly promote angiogenesis, orinhibit the activity of angiostatin promote angiogenesis.

The compounds can be, for example, antibodies, antibody fragments,enzymes, proteins, peptides, nucleic acids such as oligonucleotides, orsmall molecules. The antibodies can be, for example, monoclonal,humanized (chimeric) or polyclonal antibodies, and can be prepared, forexample, using conventional techniques. The compounds can be conjugatedto various cytotoxic agents and/or labeled compounds.

The compounds can be included in various compositions, for example,compositions suitable for intravenous, intramuscular, topical, local,intraperitoneal, or other forms of administration. They can be targetedto capillary beds by incorporating them into appropriately sizedmicroparticles or liposomes that remain lodged in capillary beds andrelease the compounds at a desired location.

Methods for promoting angiogenesis can be used to introduce vasculatureto areas in a patient that can benefit from such increased vasculature.Methods for inhibiting angiogenesis can be used to treat disordersmediated by angiogenesis, for example, tumors, autoimmune disorders suchas rheumatoid arthritis, and the like. The methods involve administeringeffective amounts of suitable angiogenic or anti-angiogenic compoundsand/or compositions including the compounds to patients in need oftreatment. Effective angiogenic amounts are amounts effective to promoteangiogenesis, and effective anti-angiogenic amounts are amountseffective to inhibit at least a significant amount of the angiogenesisthat would otherwise occur in the absence of treatment.

Screening methods can be used to identify compounds useful in thesemethods. The screening methods can identify compounds that bind to F1ATP synthase, and, in particular, the alpha and/or beta subunits, aswell as determining the activity of the compounds once bound.Combinatorial libraries of compounds, for example, phage display peptidelibraries, small molecule libraries and oligonucleotide libraries can bescreened. Compounds that bind to F1 ATP synthase, in particular, to thealpha and/or beta subunits thereof, can be identified, for example,using affinity binding studies, or using other screening techniquesknown to those of skill in the art. The effect of the compounds oncebound to the F1 ATP synthase or alpha and/or beta subunits thereof canbe determined, for example, by evaluating the level of ATP synthesis,the proliferation of human vascular endothelial cells (HUVEC), theviability and/or growth of tumors, wound healing, Matrigel™ tubeformation and corneal pocket in mouse or rat.

Nucleic acid sequences encoding F1 ATP synthase, or the alpha and/orbeta subunits or portions thereof, and host cells transformed therewith,are also disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. Direct binding assay and Scatchard analysis ofplasminogen and angiostatin with endothelial cells. HUVEC were plated ata density of 10,000 cells/well and incubated with increasingconcentrations of ¹²⁵I-labeled-plasminogen or angiostatin. FIG. 1A.¹²⁵I-labeled plasminogen binding was concentration-dependent andsaturable with an apparent dissociation constant (Kalpha) of 158 nM and870,000 sites/cell. FIG. 1B. Binding to HUVEC with ¹²⁵I-labeledangiostatin was concentration-dependent and saturable with a Kd of 245nM and 38,000 sites/cell. Error bars represent standard deviation.

FIG. 2. Competition binding assay between plasminogen and angiostatin.HUVEC were plated at a density of 10,000 cells/well and incubated with1.0 μM ¹²⁵I-labeled plasminogen in the presence of 100-fold molar excessof unlabeled angiostatin for 1 h at 4° C. Cells were washed and theremaining radioactivity was quantified by γ-counting.

(A) Total binding of 1.0 μM ¹²⁵I-labeled plasminogen was designated as100%.

(B) Plasminogen binding is inhibited by about 80% in the presence of a25-fold molar excess of unlabeled plasminogen.

(C) Plasminogen binding was not inhibited in the presence of a 100-foldmolar excess of unlabeled angiostatin suggesting distinct binding sitesfor each on the cells.

(D) showed no inhibition of binding in the presence of a 2-fold molarexcess unlabeled plasminogen

(E) Error bars represent standard deviation.

FIGS. 3A-3D. Affinity purification of plasminogen and angiostatinbinding sites. SDS-PAGE containing membrane proteins were prepared andthen analyzed by Western blotting. Membranes were incubated in 10 mMTris-HCl, 0.15M NaCl, 0.05% NP40, pH 7.5 containing, FIG. 3A,streptavidin-alkaline phosphatase conjugate antibody, or, FIG. 3B,anti-annexin II antibody and developed using5-bromo-4-chloroindol-yl-3-phosphate nitro blue tetrazolium. Membranestained with Coomassie Brilliant blue, FIG. 3C, showing affinitypurified membrane proteins. Membrane incubated with ¹²⁵I-labeledplasminogen, FIG. 3D, showing binding to the plasminogen purifiedmembrane and not the angiostatin. Lane 1 represents protein eluted fromthe plasminogen-Sepharose column. Lane 2 represents protein eluted fromthe angiostatin-Sepharose column. The relative molecular weights ofalpha-ATP synthase and beta-ATP synthase are about 59,800 Da and about56,500 Da, respectively.

FIGS. 4A-4D. Binding of antibody directed against the alpha subunit ofATP synthase on the surface of HUVEC by flow cytometry. HUVEC wereanalyzed by FACScan Flow Cytometry. Histogram plots are shown for HUVEC(FIG. 4A) and A549 (FIG. 4B) where (-) represents cells incubated withantibody directed against the alpha subunit of ATP synthase, (---)pre-immune serum and (-) secondary antibody only. Histogram plot of A549shown in FIG. 4C are similar with (...) representing antibody incubatedwith a 5-fold molar excess alpha ATP synthase protein.

In FIG. 4D, HUVEC demonstrate specific, saturable binding of antibodiesdirected against the alpha subunit of ATP synthase. The mean relativefluorescence of HUVEC incubated with pre-immune rabbit serum subtractedfrom the mean relative fluorescence of HUVEC incubated with the samevolume of anti-alpha ATP synthase gave the mean relative fluorescenceresulting from the specific binding of antibodies directed against thealpha subunit of ATP synthase on the HUVEC surface.

FIGS. 5A-5F. Immunofluorescence microscopy of ATP-synthase on HUVECsurface. HUVEC were incubated with rabbit polyclonal anti-serum raisedagainst the alpha subunit of ATP synthase from E. coli. FIG. 5A, HUVECtinder epi-illumination showing immunofluorescent surface staining forthe alpha subunit of ATP synthase. FIG. 5B, Same field of HUVEC undervisible light.

FIG. 5C, Human dermal microvascular endothelial cells also showedimmunofluorescent surface staining for the alpha subunit of ATPsynthase. Control experiments were performed with FIG. 5D, pre-immuneserum and FIG. 5E, secondary antibody alone. FIG. 5F, HUVEC werepermeabilized by acetone fixation prior to adding antibodies for thealpha subunit of ATP synthase.

FIG. 6. Competition binding assay between angiostatin and the antibodyagainst the alpha subunit of ATP synthase from E. coli. HUVEC wereplated at a constant density of 10,000 cells/well and incubated with 0.5μM ¹²⁵I-labeled angiostatin in the presence of 1:10 dilution of antibodyagainst the alpha subunit of ATP synthase from E. coli for 1 h at 4° C.Cells were washed and remaining bound radioactivity was quantified byγ-counting. Non-specific binding was measured in the presence of excessunlabeled angiostatin and was subtracted from total binding. (A) Totalbinding of 0.5 μM ¹²⁵I-labeled angiostatin was designated as 100%. (B)Angiostatin binding is inhibited by 59% in the presence of a 1:10dilution of anti-alpha subunit ATP synthase antibody. Competitionstudies were also performed simultaneously using rabbit pre-immune serumto account for non-specific inhibition. Error bars represent standarddeviation. A 1 tailed homoscedastic t test was used for statisticalanalysis; p<0.10.

FIGS. 7A-7E. Angiostatin binding to the recombinant alpha subunit ofhuman ATP synthase. The alpha subunit of human ATP synthase was clonedand expressed in E. coli and purified using Qiagen's nickel-Sepharoseprotein purification system before dialyzing in phosphate bufferedsaline (PBS), pH 7.0. Recombinant protein was electrophoresed on 5-15%SDS-PAGE, electroblotted onto Immobilon™ membrane and incubated 18 h in10 mM Tris-HCl, 0.15M NaCl, 0.05% NP40, pH 7.5 (TSN) containing¹²⁵I-angiostatin. For competition studies, unlabeled ligand was added 4h prior to radiolabeled ligand. Blots were washed in TSN buffercontaining 0.05% Tween80 and bound radioactivity was quantified on aMolecular Dynamics PhosphorImager™.

FIG. 7A. Coomassie stain of Immobilon membrane containing the alphasubunit of human ATP synthase.

FIG. 7B. Binding of 0.5 mM ¹²⁵-labeled angiostatin.

FIG. 7C. Binding of 0.5 mM ¹²⁵I-labeled angiostatin in the presence of a250-fold molar excess of unlabeled angiostatin. Binding of angiostatinis inhibited by about 56%. FIG. 7D. Binding of 0.5 nM ¹²⁵I-labeledangiostatin in the presence of a 2500-fold molar excess of unlabeledplasminogen. Binding of angiostatin is not inhibited. FIG. 7E. Bindingof 0.5 M ¹²⁵I-labeled plasminogen to the alpha subunit of human ATPsynthase. Plasminogen did not bind to the recombinant alpha subunit ofATP synthase, however, it did bind the annexin II control (as shown inFIG. 3).

FIG. 8. Binding of antibody directed against the beta subunit of ATPsynthase on the surface of HUVEC by flow cytometry. HUVEC were analyzedby FACscan Flow Cytometry as described above and in the examples.Histogram plots are shown for HUVEC cells incubated with antibodydirected against the beta subunit of ATP synthase.

FIGS. 9(a-d) are confocal micrographs showing the co-localization of thealpha- and beta-subunits of ATP synthase on the surface of HUVEC byimmunostaining and confocal microscopy. 9 a represents non-permeabilizedHUVEC immunostained with a murine monoclonal antibody specific for thealpha-subunit of ATP synthase. 9 b represents the same cellsimmunostained with a rabbit polyclonal antiserum specific for thebeta-subunit of ATP synthase. 9 c represents composite co-localizationimages obtained by digital overlays of the above images. 9 d representsa co-localization image obtained from cells permeabilized with ethanol(100%). Representative images shown, n=26.

FIGS. 10(A-F) are confocal micrographs representing the surfacelocalization of the alpha-subunits of ATP synthase and CD31 on nonpermeabilized HUVEC by immunostaining and confocal microscopy. Confocaloptical sections were taken along the z-axis every 1.5 m. Each sectionis approximately 0.6 m in thickness. A series of z-sections from arepresentative field is shown, starting with the basal surface in PanelA and ending with the apical surface in Panel D. FIG. 10E shows the samesection in panel C, with fluorescence from the red channel only. FIG.10F shows the same section in panel C, with fluorescence from the greenchannel only.

FIG. 11 is a bar graph showing the binding of angiostatin to purifiedbovine F₁ ATP synthase. ELISA was used to determineconcentration-dependent binding of angiostatin to a constant amount ofF₁ ATP synthase. Each well was coated with 1 μg of F₁ ATP synthase priorto addition of decreasing amounts of angiostatin. Control lane (-) showsbinding of secondary antibody only. n=6 Inset, Apparent dissociationconstants (K_(d(app))) were determined from double-reciprocal plots ofthe binding data. Angiostatin bound to bovine F₁ ATP synthase with aK_(d(app)) of 12 nM.

FIG. 12 is a bar graph showing the inhibition of purified F₁ ATPsynthase by angiostatin. Purified F₁ ATP synthase activity was measuredspectrophotometrically at λ=340 nm by coupling the production of ADP tothe oxidation of NADH via the pyruvate kinase and lactate dehydrogenasereaction in which a decrease in the absorbance at λ=340 nm indicatesactive protein (dark six membered rings). Angiostatin (10 M) completelyinhibited purified F₁ ATP synthase activity (light six membered rings),comparable to a known F₁ ATP synthase inhibitor, NaN₃ (2%)(darkdiamonds) and an enzyme free control (dark triangles). Polyclonalantibodies directed against the recombinant alpha-subunit of ATPsynthase (500 g/ml) (inverted dark triangles) and beta-subunit ATPsynthase (700 g/ml) (inverted light triangles) abolished ATPaseactivity. A monoclonal antibody to the alpha-subunit of ATP synthase (25g/ml) also inhibited activity (dark squares). Control antibodies had noeffect on activity (light circles and squares). Representative datashown, n=3.

FIG. 13 is a bar graph showing the inhibition of ATP generation byangiostatin on the surface of HUVEC as measured by bioluminescentluciferase assay. ATP generation on the surface of HUVEC was inhibitedin a dose-dependent manner in the presence of increasing concentrationsof angiostatin. Representative data shown, n=3.

DETAILED DESCRIPTION OF THE INVENTION

The following description includes the best presently contemplated modeof carrying out the invention. This description is made for the purposeof illustrating the general principles of the inventions and should notbe taken in a limiting sense.

Compounds, compositions and methods for promoting or inhibitingangiogenesis are disclosed. Also disclosed are screening methods foridentifying compounds that bind to the alpha and/or beta subunits of F1ATP synthase in a manner that inhibits angiostatin binding as well ascompounds that bind in an allosteric position. Methods for determiningwhether such compounds function as agonists, antagonists, partialagonists, inverse agonists, allosteric promoters or allostericinhibitors are also disclosed.

The present invention is based on the discovery that angiostatin bindsto both the alpha and beta subunits of F1 ATP synthase and, through thisbinding, inhibits angiogenesis. The active site on ATP synthase thatshuts down ATP synthesis is small and embedded in the beta subunit.While not wishing to be bound to a particular theory, it is believedthat only small molecules will actually fit into that site, so it is notlikely that angiostatin, with a molecular weight of about 35,000, orother large molecules that have molecular weights in excess of about2000, such as antibodies to the alpha and/or beta subunits of F1 ATPsynthase, with molecular weights of about 150,000, actually bind in thatsite. Accordingly, it is believed that the effect of angiostatin andother large molecules is likely a steric effect.

Compounds that bind to the alpha and/or beta subunits of F1 ATP synthasecan inhibit the ability of angiostatin to bind to these subunits, and,accordingly, inhibit the ability of angiostatin to inhibit angiogenesis.However, the compounds, once bound, may themselves function asangiogenesis inhibitors, and mimic the function of angiostatin. Thecompounds might also function as partial agonists, inverse agonists, orantagonists.

Other compounds do not inhibit the ability of angiostatin to bind to F1ATP synthase, but have an effect on the ability of angiostatin,angiostatin agonists or angiostatin partial agonists, once bound, toinhibit angiogenesis. Such compounds are referred to herein asallosteric modulators, and, depending on their effect, as allostericpromoters or allosteric inhibitors.

DEFINITIONS

The following definitions will be helpful in understanding thecompositions and methods described herein.

As used herein, the term “angiogenesis” is defined as the generation ofnew blood vessels into a tissue or organ. The term “endothelium” means athin layer of flat epithelial cells that lines serous cavities, lymphvessels, and blood vessels.

As used herein, the term “F₁-F_(o) ATP Synthase holoenzyme” (alsoreferred to herein as “ATP synthase”) is a multi-subunit enzyme thatfunctions in ATP metabolism and is typically found in the matrix of allmitochondria. ATP synthase couples proton flux across a membrane to themetabolism of ATP. There are two major complexes that togetherconstitute the holoenzyme. The F₁ complex contains multiple subunits(α₃β₃γδε) and acts as the catalytic site for ATP synthesis, whereas themembrane-embedded F_(o) complex (multiple subunits each of a, b, and c)forms a proton channel and structural attachment for F₁ (Penefsky andCross, (1991). Structure and mechanism of FoF₁-type ATP synthases andATPases. Adv Enzymol Relat Areas Mol Biol 64, 173-214). The F_(o)complex typically forms a proton channel through the inner mitochondrialmembrane, but also forms a channel through the plasma membrane incertain cells such as endothelial cells. Passage of protons through achannel formed by the a subunits causes the ring formed by the csubunits to rotate. The catalytic F₁ complex is attached to F_(o) andfaces into the mitochondrial matrix, or into the extracellular milieu inthe case of endothelial cells. Rotation of the F_(o) c-ring is coupledto rotation of the F₁ γ-subunit. The other end of the γ-subunit resideswithin a ring formed by three α and three β subunits arranged as atrimer of α-β dimers. The α-subunits perform a structural functionwhereas the β-subunits are catalytic in ATP synthesis and hydrolysis. Asthe γ-subunit rotates, it induces a series of conformational changes inthe β-subunits caused by asymmetric protein-protein interactions betweenthe γ-subunit and each of the three β-subunits. The three β-subunits arebelieved to proceed sequentially through conformational changes thatfacilitate the binding of ADP, phosphorylation to ATP, and release ofthe nascent ATP molecule (Boyer, (1997) The ATP synthase-a splendidmolecular machine. Ann Rev Biochem 66, 717-49).

The term “angiostatin” refers to a proteolytic fragment of plasminogen,and includes at least one, and preferably, at least three kringles fromplasminogen. Angiostatin is a potent inhibitor of angiogenesis and thegrowth of tumor cell metastases (O'Reilly et al., Cell 79:315-328(1994)). All anti-angiogenic forms of angiostatin are intended to beincluded within the definition of angiostatin as used herein.

Angiostatin has a specific three dimensional conformation that isdefined by the kringle region of the plasminogen molecule. (Robbins, K.C., “The plasminogen-plasmin enzyme system” Hemostasis and Thrombosis,Basic Principles and Practice, 2nd Edition, ed. by Colman, R. W. et al.J.B. Lippincott Company, pp. 340-357, 1987). There are five such kringleregions, which are conformationally related motifs and have substantialsequence homology in the amino terminal portion of the plasminogenmolecule.

A variety of silent amino acid substitutions, additions, or deletionscan be made in the above identified kringle fragments, which do notsignificantly alter the fragments' endothelial cell inhibiting activity.Each kringle region of the angiostatin molecule contains approximately80 amino acids and contains 3 disulfide bonds. Anti-angiogenicangiostatin can include a varying amount of amino- or carboxy-terminalamino acids from the inter-kringle regions and may have some or all ofthe naturally occurring di-sulfide bonds reduced. Angiostatin may alsobe provided in an aggregate, non-refolded, recombinant form.

Angiostatin can be generated in vitro by limited proteolysis ofplasminogen, as taught by Sottrup-Jensen et al., Progress in ChemicalFibrinolysis and Thrombolysis 3:191-209 (1978), the contents of whichare hereby incorporated by reference for all purposes. This results in a38 kDa plasminogen fragment (Va179-Pro353). Angiostatin can also begenerated in vitro by reducing plasmin (Gately et al., PNAS94:10868-10872 (1997)) and in Chinese hamster ovary and humanfibrosarcoma cells (Stathakis et al., JBC 272(33) :20641-.20645 (1997)).

Angiostatin may also be produced from recombinant sources, fromgenetically altered cells implanted into animals, from tumors, and fromcell cultures as well as other sources. Angiostatin can be isolated frombody fluids including, but not limited to, serum and urine. Recombinanttechniques include gene -amplification from DNA sources using thepolymerase chain reaction (PCR), and gene amplification from RNA sourcesusing reverse transcriptase/PCR.

The term “angiostatin agonist” as used herein refers to a compound(other than angiostatin) that inhibits binding of angiostatin to thealpha and/or beta subunits of F1 ATP synthase, and that binds to thealpha and/or beta subunits of F1 ATP synthase. When so bound, thecompound inhibits angiogenesis to a level equal to or greater thanangiostatin itself.

The term “angiostatin partial agonist” as used herein refers to acompound that inhibits binding of angiostatin to the alpha and/or betasubunits of F1 ATP synthase, and that binds to the alpha and/or betasubunits of F1 ATP synthase. When so bound, the compound inhibitsangiogenesis, but to a level less than angiostatin itself.

The term “angiostatin antagonist” as used herein refers to a compoundthat inhibits binding of angiostatin to the alpha and/or beta subunitsof F1 ATP synthase, and that binds to the alpha and/or beta subunits ofF1 ATP synthase. When so bound, the compound promotes angiogenesis or,at a minimum, has little or no effect on angiogenesis, thus promotingangiogenesis by inhibiting the ability of angiostatin to inhibitangiogenesis.

The term “angiostatin allosteric promoter” as used herein refers to acompound that does not inhibit the binding of angiostatin to the alphaand/or beta subunits of F1 ATP synthase, but that binds to the alphaand/or beta subunits of F1 ATP synthase in a different position on thesesubunits. When so bound, the compound promotes the ability ofangiostatin or other angiostatin agonists or partial agonists to inhibitangiogenesis.

The term “angiostatin allosteric inhibitor” as used herein refers to acompound that does not inhibit the binding of angiostatin to the alphaand/or beta subunits of F1 ATP synthase, but that binds to the alphaand/or beta subunits of F1 ATP synthase in a different position on thesesubunits. When so bound, the compound inhibits the ability ofangiostatin or other angiostatin agonists or partial agonists to inhibitangiogenesis.

The term “angiostatin allosteric modulator” as used herein refers toangiostatin allosteric promoters and inhibitors.

The terms “a”, “an” and “the” as used herein are defined to mean “one ormore” and include the plural unless the context is inappropriate.

As employed herein, the phrase “active agent” or “active compound”refers to angiostatin agonists, antagonists, partial agonists, inverseagonists or allosteric modulators. Examples of suitable biologicallyactive compounds/agents include antibodies, antibody fragments, enzymes,peptides, nucleic acids, and small molecules.

As used herein, peptide is defined as including less than or equal to100 amino acids and protein is defined as including 100 or more aminoacids.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one having ordinaryskill in the art. Although other materials and methods similar orequivalent to those described herein can be used in the practice ortesting of the present invention, as would be apparent to practitionersin the art, the preferred methods and materials are now described.

Although the alpha and beta subunits of F1 ATP synthase are described asthe binding site for angiostatin, ATP synthase couples proton fluxacross a membrane to rotation of the gamma subunit, which in turninduces cyclical conformational changes in the catalytic beta subunit.Compounds that inhibit the rotation of the gamma subunit would also beexpected to modulate angiogenesis.

I. Methods of Inhibiting Angiogenesis

There are several methods for inhibiting angiogenesis. Angiogenesis canbe inhibited by administering an effective amount of a suitableangiostatin agonist or partial agonist (for example, antibodies,antibody fragments, and/or small molecules) to a patient in need of suchtreatment. Angiostatin allosteric promoters can also be administered,alone or in combination with the angiostatin agonists and/or partialagonists. The compounds can either inhibit angiogenesis on their own, orallosterically enhance the ability of angiostatin (or other antagonistsof F1 ATP synthase) to inhibit angiogenesis. The methods can be used totreat tumors, various autoimmune disorders, hereditary disorders, oculardisorders and other angiogenesis-mediated disorders.

The therapeutic and diagnostic methods described herein typicallyinvolve administering an effective amount of the compositions describedherein to a patient. The exact dose to be administered will varyaccording to the use of the compositions and on the age, sex andcondition of the patient, and can readily be determined by the treatingphysician. The compositions may be administered as a single dose or in acontinuous manner over a period of time. Doses may be repeated asappropriate.

The compositions and methods can be used to treat angiogenesis-mediateddisorders including hemangioma, solid tumors, leukemia, metastasis,telangiectasia, psoriasis, scleroderma, pyogenic granuloma, myocardialangiogenesis, Crohn's disease, plaque neovascularization, coronarycollaterals, cerebral collaterals, arteriovenous malformations, ischemiclimb angiogenesis, corneal diseases, rubeosis, neovascular glaucoma,diabetic retinopathy, retrolental fibroplasia, arthritis, diabeticneovascularization, macular degeneration, wound healing, peptic ulcer,Helicobacter related diseases, fractures, keloids, and vasculogenesis.Specific disorders that can be treated, and compounds and compositionsuseful in these methods, are described in more detail below.

A. Carcinomas/Tumors

Carcinomas that can be treated using the compounds, compositions andmethods described herein include colorectal carcinoma, gastriccarcinoma, signet ring type, esophageal carcinoma, intestinal type,mucinous type, pancreatic carcinoma, lung carcinoma, breast carcinoma,renal carcinoma, bladder carcinoma, prostate carcinoma, testicularcarcinoma, ovarian carcinoma, endometrial carcinoma, thyroid carcinoma,liver carcinoma, larynx carcinoma, mesothelioma, neuroendocrinecarcinomas, neuroectodermal tumors, melanoma, gliomas, neuroblastomas,sarcomas, leiomyosarcoma, MFII, fibrosarcoma, liposarcoma, MPNT,chondrosarcoma, and lymphomas.

B. Ocular Disorders Mediated by Angiogenesis

Various ocular disorders are mediated by angiogenesis, and can betreated using the compounds, compositions and methods described herein.One example of a disease mediated by angiogenesis is ocular neovasculardisease, which is characterized by invasion of new blood vessels intothe structures of the eye and is the most common cause of blindness. Inage-related macular degeneration, the associated visual problems arecaused by an ingrowth of chorioidal capillaries through defects inBruch's membrane with proliferation of fibrovascular tissue beneath theretinal pigment epithelium. Angiogenic damage is also associated withdiabetic retinopathy, retinopathy of prematurity, corneal graftrejection, neovascular glaucoma and retrolental fibroplasia. Otherdiseases associated with corneal neovascularization include, but are notlimited to, epidemic keratoconjunctivitis, Vitamin A deficiency, atopickeratitis, superior limbic keratitis, pterygium keratitis sicca,periphigoid radial keratotomy, and corneal graph rejection.

Diseases associated with retinal/choroidal neovascularization include,but are not limited to, diabetic retinopathy, macular degeneration,presumed myopia, optic pits, chronic retinal detachment, hyperviscositysyndromes, trauma and post-laser complications. Other diseases include,but are not limited to, diseases associated with rubeosis(neovascularization of the angle) and diseases caused by the abnormalproliferation of fibrovascular or fibrous tissue including all forms ofproliferative vitreoretinopathy.

C. Inflammation

The methods described herein can also be used to treatangiogenesis-mediated disorders such as various forms of arthritis,including rheumatoid arthritis. In these methods, treatment withcombinations of the compounds described herein with other agents usefulfor treating the disorders, such as cyclooxygenase-2 (COX-2) inhibitors,which are well known to those of skill in the art.

The blood vessels in the synovial lining of the joints can undergoangiogenesis. The endothelial cells form new vascular networks andrelease factors and reactive oxygen species that lead to pannus growthand cartilage destruction. These factors are believed to activelycontribute to rheumatoid arthritis and also to osteoarthritis.Chondrocyte activation by angiogenic-related factors contributes tojoint destruction, and also promotes new bone formation. The methodsdescribed herein can be used as a therapeutic intervention to preventbone destruction and new bone formation.

Pathological angiogenesis is also believed to be involved with chronicinflammation. Examples of disorders that can be treated using thecompounds, compositions and methods described herein include ulcerativecolitis, Crohn's disease, bartonellosis, and atherosclerosis.

II. Methods of Promoting Angiogenesis

It is often desirable to promote angiogenesis, particularly to assist inwound healing, or to provide vascularization to occluded vessels ororgans or tissue where insufficient vascularization exists. Compoundsthat promote angiogenesis can be used to treat conditions of vascularinsufficiency, including ischemic heart disease, peripheral vasculardisease, thromboembolic disease, stroke and vasculititis (Buerger'sdisease, Wegener's granulomatosis, and Giant Cell Arteritis). Suchcompounds can also be used at wound sites to promote healing, and atsites of transplantation and grafting (e.g., skin grafting). Spinal cordinjuries can also be expected to benefit from intervention ofvascularization.

There are several methods for promoting angiogenesis. On the cellularlevel, angiogenesis can be promoted by binding a suitable compound (forexample, antibodies, antibody fragments and/or small molecules) to thealpha or beta subunit of F1 ATP synthase in a manner that inhibits theability of angiostatin to bind to the subunits, provided that thecompound itself does not itself inhibit angiogenesis when bound to thesubunits. Some compounds do not directly inhibit angiogenesis, but blockthe ability of angiostatin to inhibit angiogenesis, indirectly promotingangiogenesis. Other compounds directly promote angiogenesis by promotingATP synthesis. These types of compounds are referred to herein asangiostatin antagonists. Angiostatin allosteric inhibitors can also beused.

The methods involve administering to a patient in need of treatmentthereof an effective, angiogenesis promoting amount of an angiostatinantagonist and/or angiostatin allosteric inhibitor. An effective,angiogenesis promoting amount of such compounds is defined herein as anamount sufficient to promote angiogenesis in a patient. The amount ofsuch compounds, and the duration of treatment, can be readily determinedby a treating physician, for example, by monitoring blood flow or othersigns of increased vascularization at a desired location in a patient.

Compounds and compositions useful in the angiogenesis inhibiting andangiogenesis promoting methods are described in more detail below.

III. Compounds for Promoting or Inhibiting Angiogenesis

Various compounds, including various antibodies, can bind to a positionon the alpha or beta subunits of F1 ATP synthase and inhibit angiostatinbinding. However, the mere fact that they inhibit angiostatin bindingdoes not determine their ultimate effect on angiogenesis. Such compoundscan act as angiostatin agonists, partial agonists, inverse agonists orantagonists.

Various compounds, including various antibodies, bind to an allostericposition on the alpha and/or beta subunits of F1 ATP synthase andexercise their effect on angiostatin (or angiostatin agonists, partialagonists, inverse agonists or antagonists) in an allosteric manner, asallosteric promoters or inhibitors. The mere fact that the compoundsbind to the alpha or beta subunits in a position that does notsignificantly interfere with angiostatin binding does not determinetheir ultimate effect on the ability of angiostatin (or an agonist,partial agonist, inverse agonist or antagonist as described above) toeffect angiogenesis.

The activity of the compounds once bound can be readily determined usingthe assays described herein. The compounds described herein are notlimited to a particular molecular weight. In some cases, large compoundssuch as antibodies can be preferred since their effect is mostly steric,and therefore will not likely inhibit the function of ATP synthasesystemically, only in vascular endothelial cells. In other cases, smallmolecules may be easier to produce in commercial quantities and may beprovided in relatively larger doses. The compounds can be largemolecules (i.e., those with a molecular weight above about 1000) orsmall molecules (i.e., those with a molecular weight below about 1000).Examples of suitable types of compounds include antibodies, antibodyfragments, enzymes, peptides and oligonucleotides.

A. Antibodies

Antibodies can be generated that bind to the alpha and/or beta subunitsof F1 ATP synthase. Polyclonal antibodies can be used, provided theiroverall effect is a desired effect (i.e., an angiogenic or ananti-angiogenic effect, as desired). However, monoclonal antibodies arepreferred. Humanized (chimeric) antibodies can be even more preferred.

Angiostatin primarily binds to the outside surface of the alpha and/orbeta subunits of F1 ATP synthase, not to the inside of the subunits. Theantibodies may not and need not bind in exactly the same way asangiostatin. Angiostatin has several potential binding portions(possibly involving the various kringles), and the antibodies likely donot include portions that mimic each of these binding portions. However,the antibodies may inhibit angiostatin binding by sterically interferingwith and/or binding to all or part of the actual angiostatin bindingsite(s).

Antibodies, in particular, monoclonal antibodies (mAbs) have beendeveloped against the alpha and/or beta subunits of F1 ATP synthase thatcan be used either to directly inhibit angiogenesis or to targetcytotoxic drugs or radioisotopic or other labels to sites ofangiogenesis. Because angiogenesis does not occur to a large extent inadults, except following tissue injury, the antibodies can be extremelyspecific. Furthermore, unlike other lines of research which haveproduced cancer cell-specific mAbs to target cytotoxic drugs to tumors,these mAbs are prepared against host antigens (i.e., the alpha and betasubunits of F1 ATP synthase). This approach has the major advantage thatgeneration of “resistant” variants of the tumor cannot occur and, intheory, one mAb can be used to treat all solid tumors. An additionaladvantage is that endothelial cells, by virtue of their vascularlocation, are very accessible to antibodies in the circulation.

Antibody Preparation

The term “antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, thatspecifically binds and recognizes an analyte (antigen, in this case thealpha and/or beta subunits of F1 ATP synthase, preferably human F1 ATPsynthase). Immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit includes atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain has avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(or “VL”) and “variable heavy chain” (or “VH”) refer to these light andheavy chains, respectively.

Antibodies exist, for example, as intact immunoglobulins or as a numberof well characterized antigen-binding fragments produced by digestionwith various peptidases. For example, pepsin digests an antibody belowthe disulfide linkages in the hinge region to produce an F(ab′)₂fragment, a dimer of Fab which itself is a light chain joined to VH-CH1by a disulfide bond. The F(ab′)₂ fragment can be reduced under mildconditions to break the disulfide linkage in the hinge region, therebyconverting the F(ab′)₂ dimer into an Fab′ monomer. The Fab′ monomer isessentially an Fab with part of the hinge region (see FundamentalImmunology, Third Edition, W. E. Paul (ed.), Raven Press, N.Y. (1993),the contents of which are hereby incorporated by reference). Whilevarious antibody fragments are defined in terms of the digestion of anintact antibody, one of ordinary skill in the art will appreciate thatsuch fragments can be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments, such as a single chain antibody, anantigen binding F(ab′)2 fragment, an antigen binding Fab′ fragment, anantigen binding Fab fragment, an antigen binding Fv fragment, a singleheavy chain or a chimeric (humanized) antibody. Such antibodies can beproduced by modifying whole antibodies or synthesized de novo usingrecombinant DNA methodologies.

The alpha and/or beta subunits of F1 ATP synthase (including fragments,derivatives, and analogs thereof) can be used as an immunogen togenerate antibodies which immunospecifically bind such immunogens. Suchantibodies include but are not limited to polyclonal antibodies,monoclonal antibodies, chimeric antibodies, single chain antibodies,antigen binding antibody fragments (e.g., Fab, Fab′, F(ab′)₂, Fv, orhypervariable regions), and mAb or Fab expression libraries. In someembodiments, polyclonal and/or monoclonal antibodies to the alpha and/orbeta subunits of F1 ATP synthase are produced. In yet other embodiments,fragments of the alpha and/or beta subunits of F1 ATP synthase that areidentified as immunogenic are used as immunogens for antibodyproduction.

Various procedures known in the art can be used to produce polyclonalantibodies. Various host animals (including, but not limited to,rabbits, mice, rats, sheep, goats, camels, and the like) can beimmunized by injection with the antigen, fragment, derivative or analog.Various adjuvants can be used to increase the immunological response,depending on the host species. Such adjuvants include, for example,Freund's adjuvant (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and other adjuvants, such as BCG (bacilleCalmette-Guerin) and Corynebacterium parvum.

Any technique that provides for the production of antibody molecules bycontinuous cell lines in culture can be used to prepare monoclonalantibodies directed toward the alpha and/or beta subunits of F1 ATPsynthase. Such techniques include, for example, the hybridoma techniqueoriginally developed by Kohler and Milstein (see, e.g., Nature256:495-97 (1975)), the trioma technique (see, e.g., Hagiwara and Yuasa,Hum. Antibodies Hybridomas 4:15-19 (1993); Hering et al., Biomed.Biochim. Acta 47:211-16 (1988)), the human B-cell hybridoma technique(see, e.g., Kozbor et al., Immunology Today 4:72 (1983)), and theEBV-hybridoma technique to produce human monoclonal antibodies (see,e.g., Cole et al., In: Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96 (1985)). Human antibodies can be used and can beobtained by using human hybridomas (see, e.g., Cote et al., Proc. Natl.Acad. Sci. USA 80:2026-30 (1983)) or by transforming human B cells withEBV virus in vitro (see, e.g., Cole et al., supra).

“Chimeric” or “humanized” antibodies (see, e.g., Morrison et al., Proc.Natl. Acad. Sci. USA 81:6851-55 (1984); Neuberger et al., Nature312:604-08 (1984); Takeda et al., Nature 314:452-54 (1985)) can also beprepared. Such chimeric antibodies are typically prepared by splicingthe non-human genes for an antibody molecule specific for antigentogether with genes from a human antibody molecule of appropriatebiological activity. It can be desirable to transfer the antigen bindingregions (e.g., Fab′, F(ab′)₂, Fab, Fv, or hypervariable regions) ofnon-human antibodies into the framework of a human antibody byrecombinant DNA techniques to produce a substantially human molecule.Methods for producing such “chimeric” molecules are generally well knownand described in, for example, U.S. Pat. Nos. 4,816,567; 4,816,397;5,693,762; and 5,712,120; PCT Patent Publications WO 87/02671 and WO90/00616; and European Patent Publication EP 239 400 (the disclosures ofwhich are incorporated by reference herein). Alternatively, a humanmonoclonal antibody or portions thereof can be identified by firstscreening a cDNA library for nucleic acid molecules that encodeantibodies that specifically bind to the alpha and/or beta subunits ofF1 ATP synthase according to the method generally set forth by Huse etal., (Science 246:1275-81 (1989)), the contents of which are herebyincorporated by reference. The nucleic acid molecule can then be clonedand amplified to obtain sequences that encode the antibody (orantigen-binding domain) of the desired specificity. Phage displaytechnology offers another technique for selecting antibodies that bindto the alpha and/or beta subunits of F1 ATP synthase, fragments,derivatives or analogs thereof. (See, e.g., International PatentPublications WO 91/17271 and WO 92/01047; Huse et al., supra.)

Techniques for producing single chain antibodies (see, e.g., U.S. Pat.Nos. 4,946,778 and 5,969,108) can also be used. An additional aspect ofthe invention utilizes the techniques described for the construction ofa Fab expression library (see, e.g., Huse et al., supra) to allow rapidand easy identification of monoclonal Fab fragments with the desiredspecificity for antigens, fragments, derivatives, or analogs thereof.

Antibodies that contain the idiotype of the molecule can be generated byknown techniques. For example, such fragments include but are notlimited to, the F(ab′)₂ fragment which can be produced by pepsindigestion of the antibody molecule, the Fab′ fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragment, theFab fragments which can be generated by treating the antibody moleculewith papain and a reducing agent, and Fv fragments. Recombinant Fvfragments can also be produced in eukaryotic cells using, for example,the methods described in U.S. Pat. No. 5,965,405 (the disclosure ofwhich is incorporated by reference herein).

Antibody screening can be accomplished by techniques known in the art(e.g., ELISA (enzyme-linked immunosorbent assay)). In one example,antibodies that recognize a specific domain of an antigen can be used toassay generated hybridomas for a product which binds to polypeptidescontaining that domain. Antibodies specific to a domain of an antigenare also provided.

Antibodies against the alpha and/or beta subunits of F1 ATP synthase(including fragments, derivatives and analogs) can be used for passiveantibody treatment, according to methods known in the art. Theantibodies can be produced as described above and can be polyclonal ormonoclonal antibodies and administered intravenously, enterally (e.g.,as an enteric coated tablet form), by aerosol, orally, transdermally,transmucosally, intrapleurally, intrathecally, or by other suitableroutes.

Small amounts of humanized antibody can be produced in a transientexpression system in CHO cells to establish that they bind to HUVECcells expressing F1 ATP synthase. Stable cell lines can then be isolatedto produce larger quantities of purified material.

The binding affinity of murine and humanized antibodies can bedetermined using the procedure described by Krause et al., Behring Inst.Mitt., 87:56-67 (1990). Briefly, antibodies can be labeled withfluorescein using fluorescein isothiocyanate (FITC), and then incubatedwith HUVEC cells for two hours on ice in PBS containing fetal calf serum(FCS) and sodium azide. The amount of fluorescence bound per cell can bedetermined in a FACScan and calibrated using standard beads. The numberof molecules of antibody that had bound per cell at each antibodyconcentration can be established and-used to generate Scatchard plots.Competition assays can be performed by FACScan quantitation of boundantibody after incubating the cells with a standard quantity of themurine antibody together with a dilution series of the humanizedvariants.

Multivalent Compounds

Multivalent compounds are defined herein as compounds that include morethan one moiety capable of being attached to the alpha, beta, delta,gamma and/or epsilon subunits of F1 ATP synthase, the a, b or c subunitson F_(o) ATP synthase and/or one or more allosteric positions on theF1/F0 ATP synthase (either on the subunits themselves or a junctionalpositions at the interface between the subunits). Preferably, at leastone moiety binds to the alpha and/or beta subunits of F1 ATP synthase oran allosteric position on the F1 ATP synthase.

In one embodiment, the multifunctional compound includes at least oneprotein and/or peptide chain. Alternatively, the compound can includesmall molecules with a plurality of moieties with bind properties asdescribed above.

High Throughput Screening Methods for mAb Libraries

High throughput monoclonal antibody assays can be used to determine thebinding affinities of the antibodies to the subunits, and also identifywhich antibodies act as agonists, partial agonists, inverse agonists,antagonists and allosteric modulators of F1 ATP synthase, in particular,to the alpha and/or beta subunits thereof. The assays can evaluate, forexample, increased or decreased ATP levels or the degree of cellularproliferation. Suitable assays are described, for example, in theExamples. Similar high throughput assays can be used to evaluate theproperties of small molecule libraries.

Similar screening methods can be used to identify other classes ofcompounds useful in the methods described herein. Combinatoriallibraries of compounds, for example, phage display peptide libraries,small molecule libraries and oligonucleotide libraries can be screened.Compounds that bind to the alpha and/or beta subunits of F1 ATP synthasecan be identified, for example, using competitive binding studies. Theeffect of the compounds once bound to the F1 ATP synthase can bedetermined, for example, by evaluating the level of ATP synthesis, theproliferation of human vascular endothelial cells (HUVEC) and/or theviability and/or growth of tumors.

Antibody/Drug Conjugates

Antibodies raised against the alpha and/or beta subunits of ATPsynthase, and, in particular, monoclonal antibodies, can be conjugatedto a drug. The drug/antibody complex can then be administered to apatient, and the antibody will bind to the ATP synthase in a manner thatdelivers a relatively high concentration of the drug to the desiredtissue or organ. In some embodiments, the binding of the drug to theantibody is in a biodegradable linkage, so that the drug is releasedover time. In other embodiments, the drug remains attached to theantibody.

Anti-cancer drugs are an example of drugs that can be conjugated to theantibodies. For example, the antibodies can be conjugated with QFA,which is an antifolate, or with calicheamycin, adriaicin, bleomycin orvincamycin, which are anti-tumor antibiotics that cleave thedouble-stranded DNA of tumor cells. Additional tumor-treating compoundsthat can be coupled to the antibodies include BCNU, streptozoicin,vincristine, ricin, radioisotopes, and 5-fluorouracil and otheranti-cancer nucleosides.

In vivo xenograft studies can be used to show that tumor inhibition withlimited normal tissue damage can be obtained with antibodies conjugatedto these anti-cancer drugs. The antibody/drug conjugates can be used totarget compounds directly to tumors that might otherwise be too toxicwhen administered systemically.

The conjugates are most advantageously used in combination with targeteddrug delivery methods, for example, by placing the compounds inliposomes or other microparticles of an appropriate size such that theylodge in capillary beds around tumors and release the compounds at thetumor site. Alternatively the compounds can be injected directly into oraround the site of a tumor, for example, via injection or catheterdelivery. Such methods minimize any undesirable systemic effects.

Oligonucleotides with free, reactive hydroxy, amine, carboxy or thiolgroups at either the 3′ or 5′ end can be conjugated to free reactivegroups on antibodies using conventional coupling chemistry, for example,using heterobifunctional reagents such as SPDP. The 3′ or 5′ end of theoligonucleotide can be enzymatically labeled, for example, with ³²P astracer for DNA. The final product can be tested for cell-bindingactivity and protein and bound oligonucleotide concentrations. Dependingon the activity of the oligonucleotides, the conjugates can be used fortherapeutic or diagnostic purposes.

The antibodies (or other compounds that bind to the alpha and/or betasubunits of F1 ATP synthase) can be conjugated with photosensitizerssuch as porphyrins and used in targeted photodynamic therapy. After thecompositions are administered and allowed to bind to the F1 ATP synthasein vascular cells, the photodynamic therapy can be conducted byirradiation with light at a suitable wavelength for a suitable amount oftime.

Antibodies that bind to the alpha and/or beta subunits of F1 ATPsynthase can also be covalently or ionically coupled to various markers,and used to detect the presence of tumors. This generally involvesadministering a suitable amount of the antibody to the patient, waitingfor the antibody to bind to the F1 ATP synthase at or around a tumorsite, and detecting the marker. Suitable markers are well known to thoseof skill in the art, and include for example, radioisotopic labels,fluorescent labels and the like, and detection methods for these markersare also well known to those of skill in the art. Examples of suitabledetection techniques include positron emission tomography,autoradiography, flow cytometry, radioreceptor binding assays, andimmunohistochemistry.

Generally, a background concentration of the compounds will be observedin locations throughout the body. However, a higher, detectableconcentration will be observed in locations where a tumor is present.The label can be detected, and, accordingly, the tumors can be detected.

B. Small Molecules

As used herein, small molecules are defined as molecules with molecularweights below about 2000, except in the case of oligonucleotides thatcan be considered small molecules if their molecular weight is less thanabout 10,000 (about 30mer or less). Many companies currently generatelibraries of small molecules, and high throughput screening methods forevaluating small molecule libraries to identify compounds that bindparticular receptors are well known to those of skill in the art.Combinatorial libraries of small molecules can be screened and suitablecompounds for use in the methods described herein can be identifiedusing routine experimentation. One example of a suitable small moleculelibrary is a phage display library. Another such library is a libraryincluding random oligonucleotides, typically with sizes less than about100mers. The SELEX process can be used to screen such oligonucleotidelibraries (including DNA, RNA and other types of genetic material, andalso including natural and non-natural base pairs) for compounds thathave suitable binding properties, and other assays can be used todetermine the effect of the compounds on angiogenesis.

The SELEX method is described in U.S. Pat. No. 5,270,163 to Gold et al.Briefly, a candidate mixture of single stranded nucleic acids withregions of randomized sequence can be contacted with the alpha and/orbeta subunits of F1 ATP synthase and those nucleic acids having anincreased affinity to the subunits can be partitioned from the remainderof the candidate mixture. The partitioned nucleic acids can be amplifiedto yield a ligand enriched mixture.

C. Peptide Phage Display Libraries

One technique that is useful for identifying peptides that bind to F1ATP synthase or the alpha and/or beta subunits thereof is phage-displaytechnology, as described, for example, in Phage Display of Peptides andProteins: A Laboratory Manual; Edited by Brian K. Kay et al. AcademicPress San Diego, 1996, the contents of which are hereby incorporated byreference for all purposes.

Phage peptide libraries typically include numerous different phageclones, each expressing a different peptide, encoded in asingle-stranded DNA genome as an insert in one of the coat proteins. Inan ideal phage library the number of individual clones would be 20^(n),where “n” equals the number of residues that make up the random peptidesencoded by the phage. For example, if a phage library was screened for aseven residue peptide, the library in theory would contain 20⁷ (or1.28×10⁹) possible 7-residue sequences. Therefore, a 7-mer peptidelibrary should contain approximately 10⁹ individual phage.

Methods for preparing libraries containing diverse populations ofvarious types of molecules such as antibodies, peptides, polypeptides,proteins, and fragments thereof are known in the art and arecommercially available (see, for example, Ecker and Crooke,Biotechnology 13:351-360 (1995), and the references cited therein, thecontents of each of which is incorporated herein by reference for allpurposes). One example of a suitable phage display library is thePh.D.-7 phage display library (New England BioLabs Cat #8100), acombinatorial library consisting of random peptide 7-mers. The Ph.D.-7phage display library consists of linear 7-mer peptides fused to thepIII coat protein of M13 via a Gly-Gly-Gly-Ser flexible linker. Thelibrary contains 2.8×10⁹ independent clones and is useful foridentifying targets requiring binding elements concentrated in a shortstretch of amino acids.

Phage clones displaying peptides that are able to bind to F1 ATPsynthase or subunits thereof are selected from the library. Thesequences of the inserted peptides are deduced from the DNA sequences ofthe phage clones. This approach is particularly desirable because noprior knowledge of the primary sequence of the target protein isnecessary, epitopes represented within the target, either by a linearsequence of amino acids (linear epitope) or by the spatial juxtapositionof amino acids distant from each other within the primary sequence(conformational epitope) are both identifiable, and peptidic mimotopesof epitopes derived from non-proteinaceous molecules such as lipids andcarbohydrate moieties can also be generated.

A library of phage displaying potential binding peptides can beincubated with immobilized F1 ATP synthase, or the alpha and/or betasubunits thereof, to select clones encoding recombinant peptides thatspecifically bind the immobilized ATP synthase or subunits thereof. Thephages can be amplified after various rounds of biopanning (binding tothe immobilized F1 ATP synthase or alpha and/or beta subunits thereof)and individual viral plaques, each expressing a different recombinantprotein, or binding peptide, can then be expanded to produce sufficientamounts of peptides to perform a binding assay.

Phage selection can be conducted according to methods known in the artand according to manufacturers' recommendations. The “target” proteins,F1 ATP synthase and/or the alpha and/or beta subunits thereof, can becoated overnight onto high binding plastic plates or tubes in humidifiedcontainers. In a first round of panning, approximately 2×10¹¹ phage canbe incubated on the protein-coated plate for 60 minutes at roomtemperature while rocking gently. The plates can then be washed usingstandard wash solutions. The binding phage can then be collected andamplified following elution using the target protein. Secondary andtertiary pannings can be performed as necessary.

Following the last screening, individual colonies of phage-infectedbacteria can be picked at random, the phage DNA isolated and thensubjected to dideoxy sequencing. The sequence of the displayed peptidescan be deduced from the DNA sequence.

IV. Compositions

Therapeutic, prophylactic and diagnostic compositions containing thecompounds described herein typically include one or more activecompounds together with a pharmaceutically acceptable excipient, diluentor carrier for in vivo use. Such compositions can be readily prepared bymixing the active compound(s) with the appropriate excipient, diluent orcarrier.

Any suitable dosage may be administered. The type ofangiogenesis-mediated disorder to be treated (cancer, rheumatoidarthritis, and the like), the compound, the carrier and the amount willvary widely depending on body weight, the severity of the conditionbeing treated and other factors that can be readily evaluated by thoseof skill in the art. Generally a dosage of between about 1 milligrams(mg) per kilogram (kg) of body weight and about 100 mg per kg of bodyweight is suitable.

A dosage unit may include a single compound or mixtures thereof withother compounds or other anti-cancer agents, if the composition is usedto treat cancer, or other anti-arthritic agents, such as COX-2inhibitors, if the composition is used to treat rheumatoid arthritis.The dosage unit can also include diluents, extenders, carriers and thelike. The unit may be in solid or gel form such as pills, tablets,capsules and the like or in liquid form suitable for oral, rectal,topical, intravenous injection or parenteral administration or injectioninto or around the tumor

The compounds are typically mixed with a pharmaceutically acceptablecarrier. This carrier can be a solid or liquid and the type is generallychosen based on the type of administration being used.

The compounds can be administered via any suitable route ofadministration that is effective in the treatment of the particularangiogenesis-mediated disorder that is being treated. Treatment may beoral, rectal, topical, parenteral or intravenous administration or byinjection into the tumor and the like. The method of administering aneffective amount also varies depending on the angiogenesis-mediateddisorder being treated. It is believed that parenteral treatment byintravenous, subcutaneous, or intramuscular application of thecompounds, formulated with an appropriate carrier, additional cancerinhibiting compound or compounds or diluents to facilitateadministration, will be the preferred method of administering thecompounds.

The compounds can be incorporated into a variety of formulations fortherapeutic administration. More particularly, the compounds can beformulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, pills, powders, granules, dragees,gels, slurries, ointments, solutions, suppositories, injections,inhalants and aerosols. As such, administration of the compounds can beachieved in various ways, including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intratracheal, etc.,administration. Moreover, the compounds can be administered in a localrather than systemic manner, for example via injection of the compounddirectly into a solid tumor, often in a depot or sustained releaseformulation. In addition, the compounds can be administered in atargeted drug delivery system, for example, in a liposome coated withthe antibodies described herein. Such liposomes will be targeted to andtaken up selectively by the tumor.

In addition, the compounds can be formulated with common excipients,diluents or carriers, and compressed into tablets, or formulated aselixirs or solutions for convenient oral administration, or administeredby the intramuscular or intravenous routes. The compounds can beadministered transdermally, and can be formulated as sustained releasedosage forms and the like.

The compounds can be administered alone, in combination with each other,or they can be used in combination with other known compounds (e.g.,other anti-cancer drugs or other drugs, such as anti-inflammatories,antibiotics, corticosteroids, vitamins, etc.). For instance, thecompounds can be used in conjunctive therapy with other knownanti-angiogenic chemotherapeutic or antineoplastic agents (e.g., vincaalkaloids, antibiotics, antimetabolites, platinum coordinationcomplexes, etc.). For instance, the compounds can be used in conjunctivetherapy with a vinca alkaloid compound, such as vinblastine,vincristine, taxol, etc.; an antibiotic, such as adriamycin(doxorubicin), dactinomycin (actinomycin D), daunorubicin (daunomycin,rubidomycin), bleomycin, plicamycin (mithramycin) and mitomycin(mitomycin C), etc.; an antimetabolite, such as methotrexate, cytarabine(AraC), azauridine, azaribine, fluorodeoxyuridine, deoxycoformycin,mercaptopurine, etc.; or a platinum coordination complex, such ascisplatin (cis-DDP), carboplatin, etc. In addition, the compounds can beused in conjunctive therapy with other known anti-angiogenicchemotherapeutic or antineoplastic compounds. In pharmaceutical dosageforms, the compounds may be administered in the form of theirpharmaceutically acceptable salts, or they may also be used alone or inappropriate association, as well as in combination with otherpharmaceutically active compounds.

Suitable formulations for use in the present invention are found inRemington's Pharmaceutical Sciences (Mack Publishing Company,Philadelphia, Pa., 17th ed. (1985)), which is incorporated herein byreference. Moreover, for a brief review of methods for drug delivery,see, Langer, Science 249:1527-1533 (1990), which is incorporated hereinby reference. The pharmaceutical compositions described herein can bemanufactured in a manner that is known to those of skill in the art,i.e., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes. The following methods and excipients are merelyexemplary and are in no way limiting.

For injection, the compounds can be formulated into preparations bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives. Preferably, the compounds can be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

For oral administration, the compounds can be formulated readily bycombining with pharmaceutically acceptable carriers that are well knownin the art. Such carriers enable the compounds to be formulated astablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilicsuspensions, liquids, gels, syrups, slurries, suspensions and the like,for oral ingestion by a patient to be treated. Pharmaceuticalpreparations for oral use can be obtained by mixing the compounds with asolid excipient, optionally grinding a resulting mixture, and processingthe mixture of granules, after adding suitable auxiliaries, if desired,to obtain tablets or dragee cores. Suitable excipients are, inparticular, fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas, or from propellant-free, dry-powder inhalers. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds are preferably formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampules orin multidose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulator agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter, carbowaxes, polyethylene glycolsor other glycerides, all of which melt at body temperature, yet aresolidified at room temperature.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs. In apresently preferred embodiment, long-circulating, i.e., stealth,liposomes are employed. Such liposomes are generally described inWoodle, et al., U.S. Pat. No. 5,013,556, the contents of which arehereby incorporated by reference.

The compounds can be encapsulated in a vehicle such as liposomes thatfacilitates transfer of the bioactive molecules into the targetedtissue, as described, for example, in U.S. Pat. No. 5,879,713 to Roth etal., the contents of which are hereby incorporated by reference. Thecompounds can be targeted by selecting an encapsulating medium of anappropriate size such that the medium delivers the molecules to aparticular target. For example, encapsulating the compounds withinmicroparticles, preferably biocompatible and/or biodegradablemicroparticles, which are appropriate sized to infiltrate, but remaintrapped within, the capillary beds and alveoli of the lungs can be usedfor targeted delivery to these regions of the body followingadministration to a patient by infusion or injection.

In a preferred embodiment; the liposome or microparticle has a diameterwhich is selected to lodge in particular regions of the body. Forexample, a microparticle selected to lodge in a capillary will typicallyhave a diameter of between 10 and 100, more preferably between 10 and25, and most preferably, between 15 and 20 microns. Numerous methods areknown for preparing liposomes and microparticles of any particular sizerange. Synthetic methods for forming gel microparticles, or for formingmicroparticles from molten materials, are known, and includepolymerization in emulsion, in sprayed drops, and in separated phases.For solid materials or preformed gels, known methods include wet or drymilling or grinding, pulverization, classification by air jet or sieve,and the like.

Microparticles can be fabricated from different polymers using a varietyof different methods known to those skilled in the art. The solventevaporation technique is described, for example, in E. Mathiowitz, etal., J. Scanning Microscopy, 4, 329 (1990); L. R. Beck, et al., Fertil.Steril., 31, 545 (1979); and S. Benita, et al., J. Pharm. Sci., 73, 1721(1984). The hot-melt microencapsulation technique is described by E.Mathiowitz, et al., Reactive Polymers, 6, 275 (1987). The spray dryingtechnique is also well known to those of skill in the art. Spray dryinginvolves dissolving a suitable polymer in an appropriate solvent. Aknown amount of the compound is suspended (insoluble drugs) orco-dissolved (soluble drugs) in the polymer solution. The solution orthe dispersion is then spray-dried. Microparticles ranging between 1-10microns are obtained with a morphology which depends on the type ofpolymer used.

Microparticles made of gel-type polymers, such as alginate, can beproduced through traditional ionic gelation techniques. The polymers arefirst dissolved in an aqueous solution, mixed with barium sulfate orsome bioactive agent, and then extruded through a microdroplet formingdevice, which in some instances employs a flow of nitrogen gas to breakoff the droplet. A slowly stirred (approximately 100-170 RPM) ionichardening bath is positioned below the extruding device to catch theforming microdroplets. The microparticles are left to incubate in thebath to allow sufficient time for gelation to occur. Microparticleparticle size is controlled by using various size extruders or varyingeither the nitrogen gas or polymer solution flow rates.

Particle size can be selected according to the method of delivery whichis to be used, typically IV injection, and where appropriate, entrapmentat the site where release is desired.

Liposomes are available commercially from a variety of suppliers.Alternatively, liposomes can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811 (which is incorporated herein by reference in its entirety).For example, liposome formulations may be prepared by dissolvingappropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine,stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, andcholesterol) in an inorganic solvent that is then evaporated, leavingbehind a thin film of dried lipid on the surface of the container. Anaqueous solution of the active compound or its monophosphate,diphosphate, and/or triphosphate derivatives are then introduced intothe container. The container is then swirled by hand to free lipidmaterial from the sides of the container and to disperse lipidaggregates, thereby forming the liposomal suspension.

The monoclonal antibodies specific for F1 ATP synthase, in particular,for the alpha and/or beta subunits of F1 ATP synthase as describedherein can optionally be conjugated to liposomes and the delivery can betargeted in this manner. In addition, targeting of a marker on abnormaltumor vasculature can be employed. The targeting moiety when coupled toa toxic drug or radioisotope will act to concentrate the drug where itis needed. Ligands for tumor-associated vessel markers can also be used.For example, a cell adhesion molecule that binds to a tumor vascularelement surface marker can be employed. Liposomes and other drugdelivery systems can also be used, especially if their surface containsa ligand to direct the carrier preferentially to the tumor vasculature.Liposomes offer the added advantage of shielding the drug from mostnormal tissues. When coated with polyethylene glycol (PEG) (i.e.,stealth liposomes) to minimize uptake by phagocytes and with a tumorvasculature-specific targeting moiety, liposomes offer longer plasmahalf-lives, lower non-target tissue toxicity, and increased efficacyover non-targeted drug. Using the foregoing methods, the compounds canbe targeted to the tumor vasculature to effect control of tumorprogression or to other sites of interest (e.g., endothelial cells).

Certain organic solvents such as dimethylsulfoxide also may be employed,although usually at the cost of greater toxicity. Additionally, thecompounds may be delivered using a sustained-release system, such assemipermeable matrices of solid hydrophobic polymers containing thetherapeutic agent. Various types of sustained-release materials havebeen established and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few days up to over 100 days. Such sustainedrelease capsules typically include biodegradable polymers, such aspolylactides, polyglycolides, polycaprolactones and copolymers thereof.

Pharmaceutical compositions suitable for use in the methods describedherein include compositions wherein the active ingredients are containedin a therapeutically effective amount. The amount of compositionadministered will, of course, be dependent on the subject being treated,on the subject's weight, the severity of the affliction, the manner ofadministration and the judgment of the prescribing physician.Determination of an effective amount is well within the capability ofthose skilled in the art, especially in light of the detailed disclosureprovided herein.

Therapeutically effective dosages for the compounds described herein canbe estimated initially from cell culture assays. For example, a dose canbe formulated in animal models to achieve a circulating concentrationrange that includes the IC₅₀ as determined in cell culture (i.e., theconcentration of test compound that is lethal to 50% of a cell culture),or the IC₁₀₀ as determined in cell culture (i.e., the concentration ofcompound that is lethal to 100% of a cell culture). Such information canbe used to more accurately determine useful doses in humans. Initialdosages can also be estimated from in vivo data.

Moreover, toxicity and therapeutic efficacy of the compounds describedherein can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., by determining the LD₅₀, (thedose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index and can beexpressed as the ratio between LD₅₀ and ED₅₀. Compounds which exhibithigh therapeutic indices are preferred. The data obtained from thesecell culture assays and animal studies can be used in formulating adosage range that is not toxic for use in human. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See, e.g., Fingl et al., 1975, In: ThePharmacological Basis of Therapeutics, Ch. 1, p. 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active compound which are sufficient to maintaintherapeutic effect. Preferably, therapeutically effective serum levelswill be achieved by administering multiple doses each day. In cases oflocal administration or selective uptake,-the effective localconcentration of the drug may not be related to plasma concentration.One having skill in the art will be able to optimize therapeuticallyeffective local dosages without undue experimentation.

While the composition may be administered by routes other thanintravenously (i.v.), intraveneous administration is preferred. This isbecause the target of the therapy is primarily the proliferatingvasculature comprising the angiogenesis; and thus, administering thecomposition intravenously saturates the targeted vasculature muchquicker than if another route of administration is used. Additionally,the intravenous route allows for the possibility of further targeting tospecific tissues.

In one embodiment, a catheter is used to direct the composition directlyto the location of the target angiogenesis. For example, if tumorangiogenesis is the target of the anti-angiogenic -therapy, and if thetumor is located in the liver, then the immunoconjugate or theunconjugated antibody or a fragment thereof may be delivered into thehepatic portal vein using a catheter. In this embodiment, systemicdistribution of composition is minimized, further minimizing anypotential side effects from the antiangiogenic therapy.

V. Screening Methods

Various screening methods can be used to determine the ability ofcompounds to inhibit the binding of angiostatin to the alpha and/or betasubunits of F1 ATP synthase. In the methods described herein, compoundscan bind to a position on the alpha or beta subunits and inhibitangiostatin binding. Such compounds can act as agonists, partialagonists, inverse agonists or antagonists. The mere fact that theyinhibit angiostatin binding does not determine their ultimate effect onATP synthase.

Various other screening methods can also be used to determine theactivity of compounds bound to the alpha and/or beta subunits of F1 ATPsynthase as agonists, partial agonists, inverse agonists, antagonists,allosteric promoters and inhibitors. Examples of suitable screeningmethods include measuring ATP synthesis and measuring the cellularproliferation of human vascular endothelial cells (HUVEC).

The compounds can be evaluated using in vitro assays to determine theirbiological activity. These assays are familiar to those skilled in theart and include HUVEC and BCE proliferation assays, HUVECwound/migration assay, endothelial cell tube forming assay, CAM assay,Matrigel™ invasion assay and the rat aortic assay. The ability of acompound to inhibit or promote angiogenesis in these assays wouldindicate that the compound is either able to mimic the interaction ofangiostatin with F1 ATP synthase or the alpha and/or beta subunitsthereof, or function in an allosteric fashion.

The biological activity of the compounds may also be tested in vivo.Examples of suitable assays include the B16B16 metastasis assay or theLewis Lung Carcinoma primary tumor or metastasis assays. In suchexperiments, the activity of the compounds can be compared to that ofangiostatin if desired.

Suitable binding assays are described in more detail below.

VI. Binding Assays

ATP synthase includes two principal domains, an asymmetricmembrane-spanning Fo portion containing a proton channel and a solubleF1 portion containing three catalytic sites that cooperate in syntheticreactions. The F1 region includes subunits alpha, beta, gamma and delta.(See Elston et al., Nature 391:510 (1998).) The entire ATP synthasemolecule can be used in the present assays or a subunit thereof can beused, for example, the alpha and/or beta subunit, the angiostatinbinding domain of ATP synthase can also be used, as can a fusion proteincomprising the synthase, the subunit thereof or the angiostatin bindingdomain thereof. The Examples that follow indicate that the alpha andbeta subunits of ATP synthase are present on the plasma membrane ofendothelial cells. Further, the Examples indicate that angiostatin bindsthe alpha and/or beta subunits. The alpha and/or beta subunits presenton cellular plasma membranes may be identical to those present onmitochondrial membranes or they may represent a truncated (e.g., N- orC-terminal truncated) form thereof.

The binding assays described herein can use any such truncated forms ofthe alpha or beta subunits. Binding assays include cell-free assays inwhich ATP synthase or the alpha and/or beta or subunit thereof orangiostatin binding domain thereof (or fusion protein containing same)is incubated with a test compound (proteinaceous or non-proteinaceous)which, advantageously, bears a detectable label (e.g., a radioactive orfluorescent label). Following incubation, the ATP synthase or alphaand/or beta subunit thereof or angiostatin binding domain thereof (orfusion protein), free or bound to test compound, can be separated fromunbound test compound using any of a variety of techniques. For example,the ATP synthase (or subunit or binding domain of fusion protein) can bebound to a solid support (e.g., a plate or a column) and washed free ofunbound test compound. The amount of test compound bound to ATP synthaseor alpha and/or beta subunit thereof or angiostatin binding domainthereof (or fusion protein), is then determined, for example, using atechnique appropriate for detecting the label used (e.g., liquidscintillation counting and gamma counting in the case of a radiolabeledtest compound or by fluorometric analysis).

Binding assays can also take the form of cell-free competition bindingassays. In such assays, ATP synthase or the alpha and/or beta subunitthereof or angiostatin binding domain thereof, or fusion proteincontaining same, is incubated with a compound known to interact with F1ATP synthase, in particular, the alpha and/or beta subunits thereof,which compound, advantageously, bears a detectable label (e.g., aradioactive or fluorescent label). A test compound (proteinaceous ornon-proteinaceous) is added to the reaction and assayed for its abilityto compete with the known (labeled) compound for binding to F1 ATPsynthase or the alpha and/or beta subunit thereof or angiostatin bindingdomain thereof (or fusion protein).

Free known (labeled) compound can be separated from bound knowncompound, and the amount of bound known compound determined to assessthe ability of the test compound to compete. This assay can be formattedso as to facilitate screening of large numbers of test compounds bylinking the ATP synthase or alpha and/or beta subunit thereof orangiostatin binding domain thereof (or fusion protein), to a solidsupport so that it can be readily washed free of unbound reactants. Aplastic support, for example, a plastic plate (e.g., a 96 well dish), ispreferred. ATP synthase or alpha and/or beta subunit thereof orangiostatin binding domain thereof (or fusion protein), suitable for usein the cell-free assays described above can be isolated from naturalsources (e.g., membrane preparations) or prepared recombinantly orchemically. The ATP synthase or alpha and/or beta subunit thereof orangiostatin binding domain thereof, can be prepared as a fusion proteinusing, for example, known recombinant techniques. Preferred fusionproteins include a GST (glutathione-S-transferase) moiety, a GFP (greenfluorescent protein) moiety (useful for cellular localization studies)or a His tag (useful for affinity purification). The non-ATP synthasemoiety can be present in the fusion protein N-terminal or C-terminal tothe ATP synthase, subunit or binding domain.

As indicated above, the ATP synthase or alpha and/or beta subunitthereof or angiostatin binding domain thereof, or fusion protein, can bepresent linked to a solid support, including a plastic or glass plate orbead, a chromatographic resin (e.g., Sepharose), a filter or a membrane.Methods for attaching proteins to such supports are well known in theart and include direct chemical attachment and attachment via a bindingpair (e.g., biotin and avidin or biotin and streptavidin). Whether freeor bound to a solid support, the F1 ATP synthase or alpha and/or betasubunit thereof or angiostatin binding domain thereof, or fusionprotein, can be unlabeled or can bear a detectable label (e.g., afluorescent or radioactive label).

The binding assays also include cell-based assays in which F1 ATPsynthase or alpha and/or beta subunit thereof or angiostatin bindingdomain thereof or fusion protein, is presented on a cell surface. Cellssuitable for use in such assays include cells that naturally express F1ATP synthase and cells that have been engineered to express F1 ATPsynthase (or subunit thereof or angiostatin binding domain thereof orfusion protein comprising same). The cells can be normal or tumorigenic.Advantageously, cells expressing human ATP synthase are-used. Examplesof suitable cells include procaryotic cells (e.g., bacterial cells(e.g., E. coli)), lower eucaryotic cells, yeast cells (e g., hybrid kitsfrom Promega (CG 1945 and Y190), and the strains YPH500 and BJ5457)) andhigher eucaryotic cells (e.g., insect cells and mammalian cells (e.g.,endothelial cells, including bovine aortic endothelial cells (BAEC),bovine adrenal medulla endothelial cells (BAMEC), murine endothelialcells od-1, HUVEC or any human endothelial cell line, or cells such ashuman lung carcinoma cells (e.g., A549 cells)).

Cells can be engineered to express F1 ATP synthase (advantageously,human F1 ATP synthase or the alpha and/or beta subunit thereof orangiostatin binding domain thereof, or fusion protein that includessame) by introducing into a selected host an expression constructcomprising a sequence encoding F1 ATP synthase, or subunit thereof orangiostatin binding domain thereof or fusion protein, operably linked toa promoter. A variety of vectors and promoters can be used. For example,pET-24a(+) (Novagen) containing a T7 promoter is suitable for use inbacteria, likewise, pGEX-5X-1. Suitable yeast expression vectors includepYES2 (Invitron). Suitable baculovirus expression vectors include p2Bac(Invitron). Suitable mammalian expression vectors include pBK/CMV(Stratagene). Introduction of the construct into the host can beeffected using any of a variety of standard transfection/transformationprotocols (see Molecular Biology, A Laboratory Manual, second edition,J. Sambrook, E. F. Fritsch and T. Maniatis, Cold Spring Harbor Press,1989). Cells thus produced can be cultured using established culturetechniques suitable for the involved host. Culture conditions can beoptimized to ensure expression of the F1 ATP synthase (or subunit,binding domain or fusion protein) encoding sequence. While for thecell-based binding assays the ATP synthase (or subunit, binding domainor fusion protein) can be expressed on a host cell membrane (e.g., onthe surface of the host cell), for other purposes the encoding sequencecan be selected so as to ensure that the expression product is secretedinto the culture medium. The cell-based binding assays described hereincan be carried out by adding test compound (advantageously, bearing adetectable (e.g., radioactive or fluorescent) label), to medium in whichthe F1 ATP synthase (or alpha and/or beta subunit thereof or angiostatinbinding domain thereof or fusion protein containing same) expressingcells are cultured, incubating the test compound with the cells underconditions favorable to binding and then removing unbound test compoundand determining the amount of test compound associated with the cells.

F1 ATP synthase on a cell membrane (e.g., on the cell surface) can beidentified using techniques such as those in the Examples that follow(e.g., the cell surface can be biotin labeled and the protein followedby a fluorescent tag). Membrane associated proteins (e.g., cell surfaceproteins) can also be analyzed on a Western blot and the bands subjectedto mass spectroscopy analysis. For example, a fluorescently taggedantibody can be used, and the cells can then be probed with anotherfluorescently tagged protein. Each tag can be monitored at a differentwavelength, for example, using a confocal microscope to demonstrateco-localization.

As in the case of the cell-free assays, the cell-based assays can alsotake the form of competitive assays wherein a compound known to bind F1ATP synthase (and preferably labeled with a detectable label) isincubated with the F1 ATP synthase (or subunit thereof or angiostatinbinding domain thereof or fusion protein comprising same) expressingcells in the presence and absence of test compound. The affinity of atest compound for F1 ATP synthase can be assessed by determining theamount of known compound associated with the cells incubated in thepresence of the test compound, as compared to the amount associated withthe cells in the absence of the test compound.

The selectivity of a test compound for cell surface F1 ATP synthase, inparticular, for the alpha and/or beta subunits thereof, as compared tomitochondrial F1 ATP synthase, can be easily assessed. Compounds which,by virtue of their physicochemical properties, cannot diffuse acrosscellular membranes (and that are not natural or artificial ligands forcell transporters) can be considered selective for cell surface F1 ATPsynthase. For example, compounds that bind cell surface F1 ATP synthasebut are positively charged can thereby be prevented from diffusingacross membranes.

A test compound identified in one or more of the above-described assaysas being capable of binding to F1 ATP synthase, and, particularly, tothe alpha and/or beta subunits thereof, can, potentially, promote orinhibit angiogenesis, cellular migration, proliferation and pericellularproteolysis and, potentially, inhibit the ability of angiostatin to bindits receptor (the alpha and/or beta subunits of F1 ATP synthase). Todetermine the specific effect of any particular test compound selectedon the basis of its ability to bind the alpha and/or beta subunits of F1ATP synthase (or inhibit (competitively or non-competively) angiostatinbinding to ATP synthase), assays can be conducted to determine, forexample, the effect of various concentrations of the selected testcompound on activity, for example, cell (e.g., endothelial cell)proliferation, metabolism or cytosolic/cytoplasmic pH. (Assays can beconducted to determine the effect of test compounds on F1 ATP synthase(and F1 ATPase) activity using standard enzyme assay protocols.)

Cell proliferation can be monitored by measuring uptake of labeled basesinto cellular nucleic acids, for example, radioactively (e.g., ³H, SiC,¹⁴C), fluorescently (e.g., CYQUANT (Molecular Probes)) orcolorimetrically (e.g., BrdU (Boehringer Mannheim or MTS (Promega)).Cytosolic/cytoplasmic pH determinations can be made with a digitalimaging microscope using substrates such as BCECF(bis(carboxyethyl)-carbonyl fluorescein) (Molecular Probes, Inc.). Atest compound that reduces or replaces the concentration of angiostatinrequired to inhibit cellular proliferation or lower intracellular pH canbe expected to do so by acting as an angiostatin agonist. A testcompound that enhances cellular proliferation in the presence ofangiostatin (or functional portion thereof or functional equivalentthereof) can be expected to do so by acting as an angiostatin antagonistor allosteric inhibitor. A test compound that raises intracellular pH inthe presence of angiostatin (or functional portion thereof or functionalequivalent thereof) may do so by acting as an angiostatin antagonist.These functional assays can also be conducted in the absence ofangiostatin (i.e., test compound alone), with angiostatin (or functionalportion thereof or functional equivalent thereof) run as a separatecontrol. A test compound that, for example, modulates intracellular pHin the absence of angiostatin can be an angiostatin agonist, partialagonist, inverse agonist or antagonist.

Other types of assays that can be carried out to determine the effect ofa test compound on angiostatin binding to F1 ATP synthase include theLewis Lung Carcinoma assay (O'Reilly et al., Cell 79:315 (1994)) andextracellular migration assays (Boyden Chamber assay: Kleinman et al.,Biochemistry 25:312 (1986) and Albini et al., Can. Res. 47:3239 (1987)).Das et al. (J. Exp. Med. 180:273 (1994)) have reported the presence ofthe beta subunit of H+ transporting F1 ATP synthase on the plasmamembrane of human tumor cell lines. The present demonstration of thealpha and/or beta subunit of F1 ATP synthase on plasma membranes, andthe binding thereto by angiostatin, indicates that angiostatin may bedirectly involved in effecting cytolysis, for example, of tumor cells.The binding of angiostatin to its receptor may result in the transportof protons across plasma membranes and into cells with the result beingcytolysis by osmotic shock.

Accordingly, the methods permit the screening of compounds for theirability to modulate the effect of angiostatin on proton pumping thatresults from the binding of angiostatin to F1 ATP synthase, inparticular, to the alpha and/or beta subunits thereof. In one suchassay, cells that express F1 ATP synthase (or the alpha and/or betasubunit or portion thereof) are incubated with the test compound in thepresence of angiostatin (or functional portion thereof or functionalequivalent thereof) and the influx of protons into the cells determinedand compared to the influx of protons observed in the absence of thetest compound. Compounds that reduce the concentration of angiostatin(or functional portion thereof or functional equivalent thereof)necessary to effect a particular level of proton influx can be expectedto do so by acting as a angiostatin agonist. Compounds that reduce theamount of angiostatin-induced proton pumping observed can be expected todo so by acting as an angiostatin antagonist. The amount of protonpumping can be determined using any of a variety of approaches,including using cells preloaded with a pH sensitive reporter andmonitoring the effect of the test compound on the reporter. For example,BCECF can be used to measure pH (Misra et al., Biochem. J. 309:151(1995)). Alternatively, the effect of a test compound on proton pumpingcan be determined by monitoring cell lysis using, for example, achromium 51 release assay (McManus et al., Exper. Lung Res. 15:849(1989); Zucker et al., Res. Comm. Chem. Path. Pharm. 39:321 (1983)).

In addition to the various approaches described above, assays can alsobe designed so as to be monitorable colorometrically or usingtime-resolved fluorescence.

In another embodiment, the invention relates to compounds identifiedusing the above-described assays as being capable of binding to F1 ATPsynthase (and/or inhibiting angiostatin from binding to F1 ATP synthase(competively or non-competitively) and/or modulating the angiostatineffects on cellular bioactivities and/or modulating F1 ATP synthaseactivity. Such compounds can include novel small molecules (e.g.,organic compounds (for example, organic compounds less than 500Daltons), and novel polypeptides, oligonucleotides, as well as novelnatural products (preferably in isolated form) (including alkyloids,tannins, glycosides, lipids, carbohydrates and the like). Compounds thatbind to the alpha and/or beta subunits of F1 ATP synthase can be used toinhibit angiogenesis, for example, in tumor bearing patients and inpatients suffering from vascular related retinopathies (includingdiabetic) and Terigium.

In one embodiment, the screens first determine the binding affinity ofthe compound for the target, then look at the biochemical activity ofthe compounds, then look at cell-based or in vivo activity. An analysisof angiostatin binding or inhibition thereof can then be performed tomap possible binding sites or for a compulsive validation, but are in noway necessary to develop an effective anti-angiogenic drug.

There are two presently preferred screening assays, each used to used toscreen peptide phage display libraries. In one embodiment, purified F₁complex is coated onto high binding plastic surfaces of microtiterplates or Nunc-Immuno™ Tubes. Nonspecific protein binding surfaces areblocked by incubation with a blocking solution containing BSA, milk, oranother complex protein mixture. Unbound F₁ and excess block are washedaway and replaced by buffer. A phage library is contacted with thetarget and then unbound phage are washed away. Bound phage are eluted bytarget denaturation or by cleavage. The population of selected phage arethen contacted with bacterial cells to achieve phage infection of thesecells. Infected cultures are harvested after several hours of infectionand phage recovered by centrifugation. Such phage are then used as inputfor an additional round of selection as described above. Typically threeto five rounds of infection are performed to obtain a highly enrichedsubpopulation of the original library with affinity for F₁ complex.Individual phage are isolated by plaque assay of limiting dilutions andthen characterized as individual species. Such purified phage speciesmay be subjected to DNA sequencing in order to infer the sequence oftheir encoded peptides or antibodies. In addition, the phage may beassayed for their affinity to the purified F₁ complex or for theirability to inhibit F₁ activity. Alternatively, the peptides may beprepared as synthetic chemicals using solid phase peptide synthesis, ormay be expressed recombinantly as fusion proteins, and then assayed ineither of these forms.

In a second embodiment, the target is screened as a native enzymecomplex on the surface of endothelial cells. Cultured endothelial cellsare prepared in chamber slides and then blocked with BSA or milk asdescribed above. The phage library is contacted with the target and thenunbound phage are washed away. At this point, many phage speciesremained bound to the cell surface, with only a minority actually boundto the native F₁ complex. The phage of interest are selectively labeledby a biotinylation reaction mediated by horse radish peroxidase (HRP)that is tethered to an F₁ subunit-specific antibody. Thus, ananti-α-subunit monoclonal antibody is prepared as an HRP conjugate andthen contacted with the endothelial cells bearing specifically-boundphage. Unbound antibodies are removed by washing. The remainingantibodies are thus bound in close proximity to the phage that are boundto F₁, but not to phage binding other cell surface targets. Phage inproximity to the HRP conjugate are then biotinylated by addingbiotin-tyramine and hydrogen peroxide according to the method of Osbournet al. (Osbourn et al., (1998). Pathfinder selection: in situ isolationof novel antibodies. Immunotechnology 3, 293-302; Osbourn et al., (1998)Directed selection of MIP-1 alpha neutralizing CCR5 antibodies from aphage display human antibody library. Nat Biotechnol 16, 778-81.). Allphage are eluted as described above and the biotinylated phage are thenrecovered by passage over streptavidin beads (Dynal Biotech, New York).Active phage are then recovered by infection and amplified as above.

In both types of screens, we typically use high complexity libraries forour initial screens. Once peptide sequences are obtained, consensussequences are sought in multiple sequence alignments and efforts aremade to determine a structure-activity relationship in which theactivity may be binding affinity or ability to inhibit the enzymaticactivity. The consensus information is then used to construct a focusedmotif library that is rescreened as described above to derive highaffinity ligands with high potential for modulating bioactivity of thetarget.

The compounds identified in accordance with the above assays can beformulated as pharmaceutical compositions.

VII. Kits

Kits suitable for conducting the assays described herein can beprepared. Such kits can include F1 ATP synthase or the alpha and/or betaor other subunits thereof, or angiostatin binding domain thereof, orfusion protein comprising same, and/or angiostatin. These components canbear a detectable label. The kit can include an ATP synthase-specific orangiostatin-specific antibody. Plasminogen can also be present.

The kit can include any of the above components disposed within one ormore container means. The kit can further include ancillary reagents(e.g., buffers) for use in the assays. Diagnostic methods based on theassays for binding angiostatin to ATP synthase can be used to identifypatients suffering from angiogenesis-mediated disorders. Thedemonstration that ATP synthase is the angiostatin binding protein, andthe resulting availability of methods of identifying agents that can beused to modulate the effects of angiostatin, make it possible todetermine which individuals will likely be responsive to particulartherapeutic strategies. Treatment strategies for individuals sufferingfrom angiogenesis-mediated disorders can be designed more effectivelyand with greater predictability of a successful result. Thus, for agiven angiogenesis-mediated disorder that is of polygenic(non-Mendelian) origin, one would select that genotype that isimplicated not only in the disease, but also in that variant of thedisease that is associated with abnormal angiogenesis and proceed toscreen, via a diagnostic procedure, all future patients having the samegenotype in order to choose that therapeutic strategy most associatedwith a successful outcome or least associated with a toxic side effect,for that genotype.

The present invention will be better understood with reference to thefollowing non-limiting examples.

EXAMPLE 1 Angiostatin Binds ATP Synthase on the Surface of HumanEndothelial Cells

Summary: Angiostatin, a proteolytic fragment of plasminogen, is a potentantagonist of angiogenesis and an inhibitor of endothelial cellmigration and proliferation. To determine whether the mechanism by whichangiostatin inhibits endothelial cell migration and/or proliferationinvolves binding to cell surface plasminogen receptors, the bindingproteins for plasminogen and angiostatin were isolated from humanumbilical vein endothelial cells. Binding studies demonstrated thatplasminogen and angiostatin bound in a concentration-dependent,saturable manner. Plasminogen binding was unaffected by a 100-fold molarexcess of angiostatin, indicating the presence of a distinct angiostatinbinding site. The finding was confirmed by ligand blot analysisof-isolated human umbilical vein endothelial cell plasma membranefractions, which demonstrated that plasminogen bound to a 44-kDaprotein, whereas angiostatin bound to a 55-kDa species. Amino-terminalsequencing coupled with peptide mass fingerprinting and immunologicanalyses identified the plasminogen binding protein as annexin II andthe angiostatin binding protein as the alpha/beta-subunits of ATPsynthase. The presence of this protein on the cell surface was confirmedby flow cytometry and immunofluorescence analysis. Angiostatin alsobound to the recombinant alpha-subunit of human ATP synthase, and thisbinding was not inhibited by a 2,500-fold molar excess of plasminogen.Angiostatin's antiproliferative effect on endothelial cells wasinhibited by as much as 90% in the presence of anti-alpha-subunit ATPsynthase antibody. Binding of angiostatin to the alpha/beta-subunits ofATP synthase on the cell surface may mediate its antiangiogenic effectsand the down-regulation of endothelial cell proliferation and migration.

Tumor growth requires the continuous and persistent generation of bloodvessels. If this angiogenesis is prevented, tumor growth is dramaticallyimpaired and the tumor size is restricted. Endogenous angiogenicinhibitors therefore are likely to play an important role in tumordevelopment. Angiostatin, a proteolytic fragment of plasminogen, is apotent inhibitor of angiogenesis and the growth of tumor cell metastases(O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A.,Moses, M., Lane, W. S., Cao, Y., Sage, E. H. & Folkman, J. (1994) Cell79, 315-328). Angiostatin can be generated in vitro by limitedproteolysis of plasminogen (Sottrup-Jensen, L., Claeys, H., Zajdel, M.,Petersen, T. E. & Magnusson, S. (1978) Prog. Chem. FibrinolysisThrombolysis 3, 191-209), resulting in a 38-kDa plasminogen fragmentcontaining kringles 1-3. Although the enzymatic mechanism by whichangiostatin is generated in vivo is unknown, recent studies havedemonstrated that the cleavage of plasminogen to yield angiostatin canbe catalyzed by a serine proteinase (Gately, S., Twardowski, P., Stack,M. S., Patrick, M., Boggio, L., Cundiff, D. L., Schnaper, H. W.,Madison, L., Volpert, O., Bouck, N., et al. (1996) Cancer Res. 36,4887-4890), a macrophage metalloelastase (Dong, Z., Kumar, R., Yang, X.& Fidler, I. J. (1997) Cell 88, 801-810), and matrix metalloproteinase 3(MMP-3 or stomelysins 1) (Lijnen, H. R., Ugwu, F. & Collen, D. (1998)Biochemistry 37, 4699-4702). Generation of angiostatin from reduction ofplasmin also has been shown in vitro with human prostate carcinoma cells(Gately, S., Twardowski, P., Stack, M. S., Cundiff, D., Grella, D.,Castellino, F. J., Enghild, J., Kwaan, H. C., Lee, F., Kramer, R. A., etal. (1997) Proc. Natl. Acad. Sci. USA 94, 10868-10872), Chinese hamsterovary cells (Stathakis, P., Fitzgerald, M., Matthias, L. J., Chesterman,C. N. & Hogg, P. J. (1997) J. Biol. Chem. 272, 20641-20645), and humanfibrosarcoma cells (Stathakis, P., Fitzgerald, M., Matthias, L. J.,Chesterman, C. N. & Hogg, P. J. (1997) J. Biol. Chem. 272, 20641-20645).Additional studies demonstrated suppression of primary tumor growth inmice injected with purified angiostatin, with evidence of increasedtumor-specific apoptosis (O'Reilly, M. S., Holmgren, L., Chen, C. &Folkman, J. (1996) Nat. Med. 2, 689-692). The antiproliferative effectof angiostatin also may result from inhibition of cell cycle progression(Griscelli, F., Li, H., Bennaceur-Griscelli, A., Soria, J., Opolon, P.,Soria, C., Perricaudet, M., Yah, P. & Lu, H. (1998) Proc. Natl. Acad.Sci. USA 95, 6367-6372). However, little is known about the molecularmechanism(s) by which angiostatin functions to regulate endothelial cellbehavior.

Cellular receptors for plasminogen, including annexin II and actin, arefound on human umbilical vein endothelial cells (HUVEC) and are believedto function in the regulation of endothelial cell activities, includingangiogenesis (Hajjar, K. A., Harpel, P. C., Jaffe, E. A. & Nachman, R.L. (1986) J. Biol. Chem. 261, 11656-11662; Hajjar, K. A., Jacovina, A.T. & Chacko, J. (1994) J. Biol. Chem. 269, 21191-21197). Receptors forplasminogen also are expressed in high numbers on tumor cells where theyhave been identified as critical for tumor invasion. Proteins normallyfound in the cytoplasm, such as alpha-enolase (Miles, L. A., Dahlberg,C. M., Plescia, J., Felez, J., Kato, K. & Plow, E. F. (1991)Biochemistry 30, 1682-1691) and ATP synthase (Das, B., Mondragon, M. O.H., Sadeghian, M., Hatcher, V. B. & Norin, A. J. (1994) J. Exp. Med.180, 273-281), also occur on the cell surface and function to bindplasminogen or aid in lymphocyte-mediated cytotoxicity, respectively.The beta-subunit of mitochondrial ATP synthase is present on the surfaceof several tumor cell lines and may function to transport H+ across theplasma membrane, resulting in cytolysis. This finding is supported bystudies demonstrating addition of ATP synthase to cultures of tumor celllines induces membrane depolarization, changes in permeability, andeventual lysis of a variety of transformed cells (Virgilio, F. D.,Pizzo, P., Zanovello, P., Bronte, V. & Collavo, D. (1990) Immunol. Today11, 274-277; Rozengurt, E., Heppel, L. A. & Friedberg, I. (1977) J.Biol. Chem. 252, 4584-4590; Rozengurt, E. & Heppel, L. (1979) J. Biol.Chem. 254, 708-714; Chahwala, S. B. & Cantley, L. C. (1984) J. Biol.Chem. 259, 13717-13722; Saribas, A. S., Lustig, K. D., Zhang, X. K. &Weisman, G. A. (1993) Anal. Biochem. 209, 45-52; Virgilio, F. D.,Bronte, V., Collavo, D. & Zanovello, P. (1989) J. Immunol. 143,1955-1960; Zanovello, P., Bronte, V., Rosato, A., Pizzo, P. & Virgilio,F. D. (1990) J. Immunol. 145, 1545-1550). The presence of ATP synthaseon tumor cells may help explain lymphocyte-mediated destruction oftumors.

This example focused on the interaction of plasminogen and angiostatinwith HUVEC. Angiostatin did not compete for plasminogen binding to theendothelial cells, suggesting the presence of distinct binding sites foreach protein on the cell surface. Further studies identified theangiostatin binding site on HUVEC as the alpha/beta-subunits of ATPsynthase (alpha/beta-ATP synthase). Binding to alpha/beta-ATP synthasewas confirmed by using peptide mass fingerprinting, flow cytometry,immunohistochemical staining, Western blotting, competitive cellularbinding, and proliferation assays. These studies present evidence forthe identification of the alpha/beta-ATP synthase on the endothelialcell surface and imply a potential regulatory role for plasma membraneATP synthase in endothelial cell proliferation and migration.

Materials and Methods

Protein Purification. Plasminogen was purified from human plasma byaffinity chromatography and separated into isoforms 1 and 2 as described(Deutsch, D. & Mertz, E. T. (1970) Science 170, 1095-1096;Gonzalez-Gronow, M. & Robbins, K. C. (1984) Biochemistry 23, 190-194).Based on kinetic and electrophoretic analysis, all plasminogenpreparations were plasmin-free. The concentration of plasminogen wasdetermined spectrophotometrically at a wavelength of 280 nm by using anA_(t) %/1 cm value of 1.67 and a molecular mass of 92 kDa forGlu1-plasminogen (Castellino, F. J. (1981) Chem. Rev. 81, 431-436).Human plasminogen kringles 1-3 (angiostatin) were purified as described(Sottrup-Jensen, L., Claeys, H., Zajdel, M., Petersen, T. E. &Magnusson, S. (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209).The concentration of angiostatin was determined spectrophotometricallyat a wavelength of 280 nm by using an A₁ %/1 cm value of 0.8 and amolecular mass of 38 kDa (Sottrup-Jensen, L., Claeys, H., Zajdel, M.,Petersen, T. E. & Magnusson, S. (1978) Prog. Chem. FibrinolysisThrombolysis 3, 191-209). Protein endotoxin levels were <50.0 pgendotoxin/ml as assessed by Pyrotell Limulus amebocyte lysate clottingtimes (Associates of Cape Cod).

Cell Culture. Primary HUVEC were grown as described (Morales, D. E.,McGowan, K. A., Grant, D. S., Maheshwari, S., Bhartiya, D., Cid, M. C.,Kleinman, H. K. & Schnaper, H. W. (1995) Circulation 91, 755-763) in150-mm Petri dishes and retained for up to six passages. Human dermalmicrovascular endothelial cells were obtained from Clonetics (SanDiego), grown according to specifications, and retained for up to sixpassages. A549 (human lung carcinoma) cells were obtained from theAmerican Type Culture Collection and grown according to specifications.For all experiments, cells were detached by incubation with PBScontaining 2 mM EDTA, pH 7.4.

Antibody Purification. Antibody to His-tagged recombinant alpha-subunitATP synthase was generated in rabbits by intranodal injection (CovanceLaboratories, Vienna, Va.). Production bleeds were centrifuged, and theserum obtained was ammonium sulfate precipitated. The precipitate wasresuspended in PBS/0.5 M NaCl, pH 7.5, and passed over protein.A-Sepharose (Sigma), plasminogen-Sepharose, and alpha-subunit ATPsynthase-Sepharose columns (CNBr coupling, Amersham Pharmacia). Eachcolumn was eluted with 20 mM glycine, pH 2.5. Neutralized IgG fractionswere tested by immunodiffusion, ELISA, and Western blotting. Antibody tothe anti-alpha-subunit of ATP synthase showed no crossreactivity withplasminogen or other proteins by Western blot analysis.

Polyclonal antibody obtained from A. E. Senior (Rochester MedicalCenter, Rochester, N.Y.) directed against the alpha-subunit of ATPsynthase from Escherichia coli was characterized by ELISA and Westernblot analysis and showed no crossreactivity with other proteins in theF1 portion or E. coli cell membranes (Perlin, D. S. & Senior, A. E.(1985) Arch. Biochem. Biophys. 236, 603-611; Roa, R., Perlin, D. S. &Senior, A. E. (1985) Arch. Biochem. Biophys. 255, 309-315).

Binding Assays. Ligands were radioiodinated by using Iodobeads (Pierce),repurified on L-lysine-Sepharose, eluted with 100 mM ε-aminocaproicacid, and dialyzed in PBS, pH 7.0 before use in binding assays. HUVECwere plated at a density of 5,000 or 10,000 cells/well and incubatedwith increasing concentrations of ¹²⁵I-labeled ligand in mediacontaining 1% BSA for 1 h at 4° C. in 96-well plates. Wells were washed,and remaining bound radioactivity was quantified by using an LKB 1272γ-radiation counter. Nonspecific binding was measured in the presence ofexcess unlabeled ligand.

Membrane Purification. Plasma membrane extracts fromN-hydroxysuccinimide-biotin-labeled HUVEC were prepared by 300-psi Parrbomb nitrogen cavitation and ultracentrifugation (Young, T. N., Pizzo,S. V. & Stack, M. S. (1995) J. Biol. Chem. 270, 999-1002). Membraneextracts were incubated with plasminogen-Sepharose orangiostatin-Sepharose columns in an inhibitor mixture buffer (Young, T.N., Pizzo, S. V. & Stack, M. S. (1995) J. Biol. Chem. 270, 999-1002).Each Sepharose column was eluted with 50 mM Tris/100 mM ε-aminocaproicacid, pH 7.5, 50 mM Tris/1 M NaCl, pH 7.5, 50 mM Tris/7% dimethylsulfoxide, and 20 mM glycine, pH 2.5 to account for all types ofbinding. The glycine eluates were dialyzed, lyophilized, electrophoresedon 5-15% gradient SDS/PAGE (Laemmli, U. K. (1970) Nature (London) 227,680-685), and electoblotted onto Immobilon membrane (Matsudaira, P.(1987) J. Biol. Chem. 262, 10035-10038) before experiments to identifyplasminogen and angiostatin binding proteins.

Mass Spectrometer Analysis. Plasma membrane proteins were separated onSDS/PAGE gels, and the bands of interest were excised from the gels anddigested in situ with trypsin. A portion ( 1/20) of each sample wasanalyzed by matrix-assisted laser desorption ionization-MS, and theobtained mass spectrometric peptide maps were used to identify theprotein in the owl Protein database release 29.6 (Mann, M., Højrup, P. &Roepstrorff, P. (1993) Biol. Mass Spectrom. 22, 338-345; Pappin, D. J.C., Højrup, P. & Bleasby, A. J. (.1993) Curr. Biol. 3, 327-332).

Flow Cytometry. HUVEC and A549 cells were resuspended in ice-coldstaining buffer (Hanks' balanced salt solution/1% BSA/0.1% sodium azide)and incubated on ice for 30 min with either rabbit polyclonal antiserumraised against alpha-subunit ATP synthase derived from E. coli orpre-immune rabbit serum. Cells were washed with ice-cold staining bufferand pelleted in a microfuge at 4° C. This wash was repeated twice, andthe cells were resuspended in ice-cold staining buffer before incubationon ice for 30 min in the dark with goat anti-rabbit IgG conjugated tofluorescein isothiocyanate. After the final wash (as above), the cellswere pelleted and fixed in 10% neutral buffered formalin at a density of1×106 cells/ml. Control experiments were performed by using antibodydirected against the alpha subunit of ATP synthase, which waspreincubated with a 5-fold molar excess of recombinant alpha-subunit ATPsynthase protein. The mean relative fluorescence after excitation at awavelength of 488 nm was determined for each sample on a FACScan flowcytometer (Becton-Dickenson) and analyzed with cellquest software(Becton-Dickenson).

Immunofluorescence Microscopy. HUVEC and human dermal microvascularendothelial cells were plated at 5×10⁵ cells/ml on glass coverslips andallowed to adhere overnight. Cells were incubated at 4° C. for 1 h inPBS, pH 7.0 containing 1% BSA with either rabbit polyclonal antiserumraised against the alpha subunit of ATP synthase derived from E. coli,pre-immune rabbit serum, pre-immune IgG, or anti-rabbit IgG. Cells werewashed and incubated at 4° C. for 1 h in the dark with goat anti-rabbitIgG conjugated to indocarbocyanine (Cy3) before washing and fixing in 4%paraformaldehyde. Immunofluorescence microscopy was performed by usingan Olympus BX-60 microscope (Olympus, Lake Success, N.Y.).

Cloning of the alpha-Subunit of ATP Synthase. Poly(A)+ mRNA was isolatedfrom HUVEC by using Oligotex resin (Qiagen). RNA was reverse-transcribedinto single-stranded cDNA by using AMV Reverse Transcriptase (BoehringerMannheim). The alpha subunit of ATP synthase was PCR-amplified by usingExpand High Fidelity PCR system (Boehringer Mannheim). The 1.7-kb PCRproduct was purified from a 0.8% Tris-acetate/EDTA agarose gel by usinga QIAEX II gel extraction kit. Restriction enzyme digests of the PCRfragment and vector pLE1 were carried out at 37° C. for 1 h. Bothdigests were passed over Qiaquick purification columns, then ligatedovernight at 16° C. by using T4 DNA ligase. Competent E. Coli DH5a(GIBCO/BRL) were transformed with the ligation mixture, plated on 2×Bacto-yeast tryptone (YT) agarose plates, and grown overnight at 37° C.Colonies were screened for the insert by restriction enzyme digestionand DNA sequencing.

Purification of the alpha-Subunit of ATP Synthase. Competent E. coliBL21DE3 were transformed with the pET24a(+) vector containing the alphasubunit, plated on 2× Bacto-yeast tryptone (YT) agarose, and grownovernight at 37° C. Twenty milliliters of 2× YT containing 50 mg/mlkanamycin was inoculated with one colony and grown overnight at 37° C.(200 rpm). A 1-liter culture (2× YT, 50 mg/ml kanamycin) was inoculatedwith 20 ml of the noninduced overnight culture and grown at 37° C. to anOD of 0.6 at a wavelength of 600 nm. Isopropyl thio-beta-d-galactosidasewas added to a final concentration of 1 mM and grown for an additional 3h. Cells were harvested by centrifugation at 8,000 rpm for 10 min andstored overnight at −20° C. Lysates were prepared under denaturingconditions and batch-purified by using Qiagen Ni-NTA agarose. Resultingprotein was dialyzed against PBS, pH 7.0 for use in all experiments.

Proliferation Assay. HUVEC were plated at a density of 5,000 cells/wellin media depleted of FCS overnight to allow the cells to becomequiescent. Fresh media containing FCS were added to the wells along withangiostatin at a final concentration of 0.5, 0.75, and 1.0 mM. In someexperiments antibody directed against the alpha subunit of ATP synthasederived from E. coli was also added at a dilution of 1:10. MTS/PMS[(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt/phenazine methosulfate] solution was added after 24 h, andthe absorbance of formazan was quantitated on a Thermomax plate readerat a wavelength of 490 nm according to the manufacturer's specifications(Promega). The absorbance values used to calculate the percentproliferation of the cells ranged from 0.81 for untreated, 0.60 fortreated, and 0.47 for baseline quiescent cells.

Results and Discussion

Binding of Plasminogen and Angiostatin to Endothelial Cells. Todetermine whether angiostatin blocks angiogenesis by competitiveinteraction with endothelial cell plasminogen receptors, the effects ofangiostatin on the binding of plasminogen to endothelial cells wereanalyzed. In control experiments, plasminogen bound to HUVEC in aconcentration-dependent saturable manner with an apparent dissociationconstant (Kd) of 158 nM and approximately 870,000 sites per cell (FIG.1A), comparable to values previously reported (Hajjar, K. A., Jacovina,A. T. & Chacko, J. (1994) J. Biol. Chem. 269, 21191-21197). Angiostatinalso bound to HUVEC in a concentration-dependent saturable manner with asimilar affinity (Kd of 245 nM), with approximately 38,000 sites percell (FIG. 1B). Binding studies using ¹²⁵I-labeled plasminogen and a100-fold molar excess of unlabeled angiostatin demonstrated noinhibition of plasminogen binding (FIG. 2). Similar studies wereperformed by using ¹²⁵I-labeled angiostatin. Excess unlabeledplasminogen had little or no effect on angiostatin binding (FIG. 2). Incontrast to plasminogen, binding of ¹²⁵I-labeled angiostatin to HUVEC inthe presence of 100 mM ε-aminocaproic acid was only slightly inhibited,suggesting that binding of angiostatin to endothelial cells is not alysine binding site-dependent process (data not shown). Together thesedata suggest the presence of a distinct angiostatin binding site onHUVEC.

Purification of the Angiostatin Binding Site from Endothelial Cells. Thecell surface proteins involved in binding of plasminogen or angiostatinto HUVEC were identified by subjectingN-hydroxysuccinimide-biotin-labeled HUVEC plasma membranes to affinitychromatography on plasminogen-Sepharose or angiostatin-Sepharose. Twodistinct bands were identified on Western blot analysis usingstreptavidin-alkaline phosphatase conjugate (FIG. 3A) or by Coomassiebrilliant blue stain (FIG. 3C). A companion blot, probed with anantibody to the known plasminogen receptor, annexin II, demonstratedimmunologic crossreactivity with the 44-kDa membrane protein isolatedfrom the plasminogen-Sepharose column (FIG. 3B, lane 1), but not withthe 55-kDa protein isolated from the angiostatin-Sepharose column (FIG.3B, lane 2). Ligand blot analysis of the affinity-purified plasmamembranes using ¹²⁵I-labeled plasminogen (FIG. 3D, lanes 1 and 2)demonstrated binding of plasminogen only to the 44-kDa protein and notto the 55-kDa species, providing additional evidence that HUVEC containan angiostatin binding site distinct from the known plasminogen bindingprotein, annexin II.

The proteins were separated on an SDS/PAGE gel, the band of interest wasexcised from the gel, and then digested in situ with trypsin. Then 1/20of the sample was analyzed by matrix-assisted laser desorptionionization-MS. The obtained mass spectrometric peptide map was used toidentify the alpha/beta-subunits of ATP synthase in the owl Proteindatabase release 29.6 (Mann, M., Højrup, P. & Roepstrorff, P. (1993)Biol. Mass Spectrom. 22, 338-345; Pappin, D. J. C., Højrup, P. &Bleasby, A. J. (1993) Curr. Biol. 3, 327-332). Analysis of proteinsequences of alpha and beta ATP synthase from the Institute of Biologyand Chemistry of Proteins showed 23% homology and 57% similarity.

Peptide Mass Fingerprinting of the Angiostatin Binding Site. To identifythe unique angiostatin binding site component, the affinity-purifiedbinding proteins were analyzed by amino-terminal sequencing, massspectrometer analysis, and peptide mass fingerprinting. Both the 44- and55-kDa proteins were analyzed by reduced SDS/PAGE and digested withtrypsin in situ (Matsui, N. M., Smith, D. M., Clauser, K. R., Fichmann,J., Andrews, L. E., Sullivan, C. M., Burlingame, A. L. & Epstein, L. B.(1997) Electrophoresis 18, 409-417). The resulting peptides wereextracted and the mass of approximately 30 peptides was determined byusing a Bruker Reflex matrix-assisted laser desorption ionization-timeof flight mass spectrometer, providing a unique signature by which toidentify the protein by peptide mass searches. The 55-kDa angiostatinbinding membrane protein was identified as the alpha/beta-subunits ofATP synthase (Table 1), whereas the plasminogen binding protein wasconfirmed as annexin II. Although expression of the beta-subunit of ATPsynthase has been reported on the surface of several tumor cell lines(Das, B., Mondragon, M. O. H., Sadeghian, M., Hatcher, V. B. & Norin, A.J. (1994) J. Exp. Med. 180, 273-281), evidence is provided herein forsurface expression of the alpha/beta-subunits of ATP synthase on HUVEC.TABLE 1 Bruker Reflex Matrix-assisted laser desorption ionization-timeof flight mass spectrometer analysis of 55-kDa peptides Peptide Mass(monoisotopic) Calculated Sequence Measured (Da) (Da) QMSLLLR 859.48859.495 AVDSLVPIGR 1025.58 1025.587 VGLKAPGIIPR 1119.68 1119.713TIAMDGTEGLVR 1261.40 1261.634 ISVREPMQTGIK 1357.70 1357.739 IMNVIGEPIDER1384.68 1384.702 AHGGYSVFAGVGER 1405.66 1405.674 FTQAGSEVSALLGR 1434.731434.747 TSIAIDTIINQKR 1471.81 1471.836 EAYPGDVFYLHSR 1552.71 1552.731VALVYGQMNEPPGAR 1600.79 1600.803 TGAIVDVPVGEELLGR 1623.87 1623.883LVLEVAQHLGESTVR 1649.88 1649.910 IMDPNIVGSEHYDVAR 1814.85 1814.862VLDSGAPIKIPVGPETLGR 1918.08 1918.089 AIAELGIYPAVDPLDSTSR 1986.991987.026 IMNVIGEPIDERGPIKTK 2009.10 2009.098 IPSAVGYQPTLATDMGTMQER2265.06 2265.077 EVAAFAQFGSDLDAATQQLLSR 2337.15 2337.160Binding of the Alpha-Subunit ATP Synthase Antibody to the Surface ofHUVEC by Flow Cytometry and Immunofluorescence Microscopy.

To further confirm the surface localization of the ATP synthase, HUVECwere analyzed by flow cytometry and immunoflourescence microscopy. Arabbit polyclonal antiserum raised against the alpha subunit of ATPsynthase from E. coli reacted with the cell membranes of HUVEC asdetermined by fluorescence-assisted flow cytometry (FIG. 4A). Controlflow cytometry studies were performed by using A549 cells, which areknown to express the alpha/beta-subunits of ATP synthase (Das, B.,Mondragon, M. O. H., Sadeghian, M., Hatcher, V. B. & Norin, A. J. (1994)J. Exp. Med. 180, 273-281) (FIG. 4B). A549 cells also were analyzed withanti-alpha-subunit ATP synthase antibody pre-incubated with a 5-foldmolar excess of recombinant alpha-subunit of ATP synthase protein andshowed a decreased affinity for binding (FIG. 4C). HUVEC were incubatedwith increasing concentrations of antibody to determine saturation. FIG.4D demonstrates specific, saturable binding of antibody directed againstthe alpha subunit of ATP synthase on HUVEC membranes.

Immunofluorescence microscopy of HUVEC confirmed the surface-associatedimmunoreactivity of alpha-subunit ATP synthase antibody on HUVEC cellmembranes (FIG. 5A). Control experiments were performed with secondaryantibody alone (FIG. 5D), pre-immune serum (FIG. 5E), and permeabilizedHUVEC in the presence of anti-alpha-subunit ATP synthase antibody (FIG.5F). Human dermal microvascular endothelial cells also reacted withantiserum raised against the alpha subunit of ATP synthase (FIG. 5C).

Inhibition of Angiostatin Binding in the Presence of the Antibody to theAlpha-Subunit of ATP Synthase.

The rabbit polyclonal antiserum raised against the alpha-subunit ATPsynthase blocked binding of angiostatin to HUVEC by 59%, demonstratingthat this protein functions in angiostatin binding (FIG. 6). Inaddition, ¹²⁵I-labeled angiostatin bound to purified recombinant alphasubunit from human ATP synthase (FIG. 7B), and binding was inhibited˜56% in the presence of a 250-fold molar excess of unlabeled angiostatin(FIG. 7C). Complete inhibition of binding was not obtained and may becaused in part by nonspecific binding, improper folding of therecombinant protein, or binding epitopes only found in the presence ofthe alpha/beta-heterodimer. Furthermore, binding to the recombinantalpha subunit ATP synthase was not inhibited by a 2,500-fold molarexcess of unlabeled plasminogen (FIG. 7D). ¹²⁵I-labeled plasminogen didnot bind to the recombinant alpha subunit of ATP synthase (FIG. 7E), butdid bind to annexin II (FIG. 3D).

Inhibition of Proliferation in the Presence of Antibody to theAlpha-Subunit of Human ATP Synthase.

To determine whether the antiproliferative effects of angiostatin weremediated by ATP synthase binding, cell proliferation assays wereperformed in the presence of antiserum raised against the alpha subunitof ATP synthase from E. coli. The inhibitory effects of angiostatin onHUVEC proliferation were abrogated by approximately 81% in the presenceof antibody to the alpha subunit of ATP synthase (Table 2), providingdirect evidence that angiostatin binding to the alpha subunit of ATPsynthase functions as a mechanism for inhibition of endothelial cellgrowth. These data indicated that this binding site could serve as areceptor for angiostatin. TABLE 2 The antiproliferative effect ofangiostatin is reversed by anti-alpha subunit ATP synthase antibodyPercent proliferation Concentration inhibited, ±SEM angiostatin added,Without With % μM Antibody antibody Recovery 0 0 0 0 0.5 10 ± 1.4 1 ±0.2 90 0.75 25 ± 4.2 5 ± 4.1 80 1.0 23 ± 9.0 6 ± 0.8 74

HUVEC were plated at a density of 5,000 cells/well in media containingangiostatin at a final concentration of 0.5, 0.75, and 1.0 mM.Anti-alpha-subunit ATP synthase antibody derived from E. coli was addedconcomitantly at a dilution of 1:10. MTS/PMS solution was added andabsorbance of formazan was quantitated according to the manufacturer'sspecifications. Results represent three separate experiments performedin duplicate with SEM. Percent recovery represents the ability of theanti-alpha-subunit ATP synthase antibody to block the antiproliferativeeffect of angiostatin, and thereby restore proliferation to an averageof 81% of that obtained with the control cells.

ATP synthase is composed of two functional domains termed F1 and F0. TheF1 portion contains multiple subunits (α3β3γδε) and acts as thecatalytic site for ATP synthesis, whereas the membrane-embedded F0portion is a portion channel (Penefsky, H. S. & Cross, R. L. (1991) Adv.Enzymol. Relat. Areas Mol. Biol. 64, 173-214). Isolated alpha and betasubunits bind ATP and have weak ATPase activity; however, ATP synthesisrequires all F1 sand F0 subunits (Boyer, P. D. (1997) Ann. Rev. Biochem66, 717, 749).

Endothelial cells play a strategic role within the vasculature, servingas a barrier between the intravascular compartment and the underlyingtissues, and often are exposed to hypoxic stress. Relative to other celltypes, endothelial cells are more resistant to hypoxic challenge bytheir ability to maintain a high level of intracellular ATP (Graven, K.K. & Farber, H. W. (1997) Kidney Int. 51, 426-437). It is interesting tospeculate that a plasma membrane-associated ATP synthase may produceextracellular ATP, which can diffuse back into the cell, providing anadditional, albeit limited, ATP source (Unno, N., Menconi, M. J.,Salzman, A. L., Smith, M., Hagen, S., Ge, Y., Ezzell, R. M. & Fink, M.P. (1996) Am. J. Physiol. 270, G1010-G1021; Unno, N., Menconi, M. J.,Smith, M., Hagen, S. J., Brown, D. A., Aquirre, D. E. & Fink, M. P.(1997) Surgery 121, 668-680). Angiostatin, by binding to thealpha/beta-subunits of plasma membrane-localized ATP synthase, maydisrupt this production of ATP, rendering endothelial cells morevulnerable to hypoxic challenge and eventual irreversible cell damage.In the microenvironment of a growing tumor, tissue hypoxia provides apowerful stimulus for the production of angiogenic growth factors suchas vascular endothelial growth factor, basic fibroblast growth factor,and angiopoetin. The ability of host endothelial cells to respond tothese growth factors by increased proliferation likely depends on theirability to survive hypoxic challenge. By abolishing the ability toresist low oxygen tension, angiostatin may decrease endothelial cellsurvival in the tumor microenvironment. It recently has been reportedthat angiostatin also may function by inducing endothelial cellapoptosis, providing an additional independent mechanism for theantiangiogenic action of this polypeptide (Claesson-Welsh, L., Welsh,M., Ito, N., Anand-Apte, B., Soker, S., Zetter, B., O'Reilly, M. &Folkman, J. (1998) Proc. Natl. Acad. Sci. USA 95, 5579-5583).

Binding of Antibody Directed Against the Alpha Subunit of ATP Synthaseon the Surface of HUVEC by Flow Cytometry

HUVEC cells were resuspended in ice-cold staining buffer (HBSS, 1% BSA,0.1% sodium azide) and incubated on ice for 30 min with either rabbitpolyclonal anti-serum raised against alpha subunit ATP synthase derivedfrom E. coli or pre-immune rabbit serum. Cells were washed with ice-coldstaining buffer and pelleted in a microfuge at 4° C. This wash wasrepeated twice and the cells resuspended in ice-cold staining bufferprior to incubation on ice for 30 min in the dark with goat anti-rabbitIgG conjugated to fluorescein isothiocyanate (FITC). Following the finalwash (as above), the cells were pelleted and fixed in 10% neutralbuffered formalin at a density of 1×10⁶ cells/mi. Control experimentswere performed using antibody directed against the alpha subunit of ATPsynthase which was preincubated with a 5-fold molar excess ofrecombinant alpha subunit ATP synthase protein. The mean relativefluorescence following excitation at a wavelength of 488 nm wasdetermined for each sample on a FACScan flow cytometer(Becton-Dickenson) and analyzed with CellQuest software(Becton-Dickenson). Histograms are shown in FIG. 8.

EXAMPLE 2 Demonstration that Endothelial Cell-Surface F₁-F_(o) ATPSynthase is Active in ATP Synthesis and is Inhibited by Angiostatin

As discussed herein, angiostatin blocks tumor angiogenesis in vivo. Thisexample demonstrates that F₁-F_(o) ATP synthase is the major angiostatinbinding site on the endothelial cell-surface, and this data suggeststhat ATP metabolism may play a role in the angiostatin response. Thedata also demonstrate that all components of the F₁ ATP synthasecatalytic core are present on the endothelial cell surface, where theyco-localize into discrete punctate structures. The surface-associatedenzyme is active in ATP synthesis as shown by dual label thin layerchromatography and bioluminescence assays. Both ATP synthase and ATPaseactivities of the enzyme are inhibited by angiostatin as well as byantibodies directed against the alpha and beta subunits of ATP synthasein cell-based and biochemical assays. The data suggest that angiostatininhibits vascularization by suppression of endothelial surface ATPmetabolism, which in turn may regulate vascular physiology byestablished mechanisms. Antibodies directed against subunits of ATPsynthase exhibit endothelial cell-inhibitory activities comparable tothat of angiostatin, indicating that these antibodies function asangiostatin mimetics.

This example demonstrates conclusively that all components forming thecore catalytic complex of F₁ ATP synthase are present on the externalendothelial cell surface. Moreover, this enzyme is catalyticallycompetent in the synthesis of ATP on the surface of endothelial cells.Finally, both angiostatin and antibodies against the alpha and betasubunits of ATP synthase inhibit activity of the surface-associatedenzyme and of the purified enzyme. Taken together, these data stronglysuggest that ATP synthase is the primary target for angiostatin on thesurface of endothelial cells and further suggest that cell-surface ATPmetabolism is likely to play a central role in the endothelial cellresponse to angiostatin. The ability of angiostatin or antibodies todisrupt ATP production renders endothelial cells more vulnerable tohypoxic challenge in the microenvironment of a growing tumor andadditionally alters ATP-mediated signal transduction at the endothelialcell surface.

Materials and Methods

Confocal Microscopy. HUVEC were plated in EGM-MV media at 150,000cells/ml on glass coverslips and allowed to adhere overnight. Cells wereincubated at 4° C. and washed with PBS before fixation in 2%paraformaldehyde solution. A control slide was permeabilized in 100%ethanol for 5 min at rt before fixation. All coverslips were incubatedin 5% goat serum in Dulbecco's PBS pH7.4 (Life Technologies,Gaithersburg, Md.) for 15 min and washed before co-incubation withmurine monoclonal anti-alpha ATP synthase (Molecular Probes Inc.,Eugene, Oreg.) (1:100) and rabbit polyclonal antibody raised against therecombinant human beta-subunit of ATP synthase (1:500) diluted instaining buffer (1% BSA/0.02% Tween 20/0.005M EDTA/1% goat serum/1×PBS,pH 7.4). To verify surface staining, cells were incubated with murinemonoclonal anti-CD31 (PharMingen, Los Angeles, Calif.). All cells werewashed three times and incubated for 1 hr in the dark at 4° C. with goatanti-mouse IgG AF488 F(ab′)₂ (Molecular Probes) (1:100), goatanti-rabbit IgG AF546 F(ab′)₂ (Molecular Probes) (1:200), or goatanti-mouse IgG1 AF633 (1:100) in staining buffer. After the finalwashes, cells were visualized using a Zeiss LSM-410 (Switzerland)confocal microscope at 630×.

ELISA Binding Studies. Binding studies were performed with purified F₁subunit of bovine ATP synthase (20 g/ml) passively adsorbed ontopolyvinylchloride microtiter 96-well flat bottom plates (DynexTechnologies, Inc, Chantilly, Va.) as previously described (22).Briefly, plates were coated with protein in 50 μl of 0.1M Na₂CO₃, pH9.6and incubated 2 h at 37° C. Non-specific sites were blocked byincubating with PBS, pH 7.0 containing 1% BSA for 30 min at rt. Bindingstudies were performed with increasing amounts of angiostatin (0-0.5mg/ml) added in a 50 1 final volume for 1 h at 37° C. Plates were washed(0.1% Tween20/PBS, pH 7.0) and incubated with anti-human angiostatinantibody (goat IgG) (R&D Systems, Minneapolis, Minn.) (1:200 dil)overnight at 4° C. Plates were washed and incubated with anti-goat IgGperoxidase conjugate (Sigma, St. Louis, Mo.) (1:7500 dil) for 1 h at 37°C. Plates were washed and 150 μl of OPD substrate was added to each welland the reaction stopped with 25 μl of H₂SO₄ (25%), before monitoringthe absorbance at λ=492 nm in a Molecular Devices SpectraMax Plus-384plate reader. Control studies were performed in the absence ofangiostatin to detect any non-specific binding of secondary proteins.Isotype specific controls were performed using a goat anti-fibronectinantibody (Sigma).

Purification of Bovine Heart F₁ ATP Synthase and F₁ Activity Assay.Fresh bovine heart mitochondria were obtained as previously described(23) and sonicated to yield sub-mitochondrial particles (24). The F₁portion was separated from membrane-bound F_(o) by chloroformextraction. The aqueous layer was centrifuged at 105,000×g to removeparticulate matter before purifying over an S300 gel filtration column.The purified F₁ ATP synthase only exhibits activity in the reversereaction (ATPase), because the forward reaction (ATP synthase) requiresthe F₁-F_(o) holoenzyme correctly assembled into a membrane across whicha proton gradient exists. ATPase activity was measuredspectrophotometrically by monitoring at λ=340 nm by coupling theproduction of ADP to the oxidation of NADH via pyruvate kinase andlactate dehydrogenase reactions as described in Zheng,& Ramirez, BiochemBiophys Res Commun 261(25):499-503 (1999).

Cell-surface ATP Assay. Quiescent, confluent HUVEC in 24 well plateswere washed and equilibrated into DMEM/F-12 (HAM) 1:1 medium (LifeTechnologies) containing 10 mM potassium phosphate. Cells were treatedwith (1 μM) angiostatin or (0.1 mg/mL) oligomycin for 1 hr at 37° C. Allcells were then incubated with 0.1 Ci ³²P and 0.1 Ci (50 M) [2,8-³H]-ADP(NEN Life Sciences) for 1 min. Supernatants were removed and centrifugedbefore assaying for ATP production by thin layer chromatography (TLC) orby firefly luciferase assay.

ATP Generation by Bioluminescent Luciferase Assay. Aliquots (50 μl ) ofcellular supernatants from cell-surface ATP assays were analyzed usingthe ATP bioluminescence assay kit (Sigma Chemical, St. Louis, Mo.). Inthis firefly luciferin-luciferase reaction, only ATP is readily detectedsince the enzymatic reaction of firefly luciferase to oxidize luciferinis specific for ATP relative to all other nucleotides. Samples wereinjected with the ATP assay mixture and recordings were made in aLuminoskanRS (LabSystems, Franklin, Mass.) over a 10 s period. Theresponse in a given sample or standard was quantified as area under thepeak of the response and averaged for duplicate determinations. Data areexpressed as picomoles of ATP produced per cell based on standardsdetermined under the same conditions with each experiment. Angiostatinwas prepared as described in Sottrup-Jensen, L., Claeys, H., Zajdel, M.,Petersen, T. E. & Magnusson, S. (1978) Progress in Chemical Fibrinolysisand Thrombolysis (Raven Press, New York). Angiostatin did not interferewith the luciferin-luciferase assay, indicating that the effects ofangiostatin are not an assay artifact (data not shown).

Dual Label Radioactive Thin Layer Chromatography of ATP. Supernatantswere obtained as described above. Cell pellets were obtained afterwashing wells with 1.0 ml media (as described above) and lysing with 1NNaOH (100 μl). Aliquots of supernatant (3 l) and cell pellet (10 μl)were applied to microcrystalline cellulose PEI plates (Anal. Tech.,Newark, Del.) along with an authentic [³²P]ATP standard (0.025 Ci).Plates were developed in 1.4 M LiCl for a distance of 15 cm. Dried spotscontaining [³²P]ATP, [³H]ATP, and [³H]ADP were detected by sodium iodideand phosphoimaging on a STORM 850 (Molecular Dynamics, Sunnyvale,Calif.). Areas corresponding in R_(f) value to co-chromatographedauthentic [³²P]ATP standard were scraped off the plate and theirradioactivity determined in a liquid scintillation analyzer (PackardBioScience Co., Meridan, Conn.). No ATP was detectable by TLC in the[³H]ADP preparation used for all experiments.

Cell Proliferation Assay. HUVEC were plated at a density of 5000cells/well in media depleted of fetal calf serum (FCS) overnight toallow the cells to become quiescent. Fresh media containing 5% FCS, 10ng/ml basic fibroblast growth factor (bFGF) and 3 ng/ml vascularendothelial growth factor (VEGF) were added to the wells along withangiostatin (1.0 μM), antibody directed against the recombinant humanalpha-subunit of ATP synthase (1:10 dil), antibody directed against therecombinant human beta-subunit of ATP synthase (1:10 dil), pre-immuneserum (1:10 dil) or cycloheximide (10 g/ml). Cell density was measuredafter 24 hrs using CyQUANT Cell Proliferation Assay Kit (MolecularProbes) in a fluorometric plate reader (Molecular Devices). Theabsorbance values used to calculate the percent proliferation of thecells ranged from 1.24 or 1.00 for treated and 2.57 for untreated.

Results: Alpha and Beta-Subunits of ATP Synthase Co-Localize on theSurface of HUVEC.

Extensive co-localization of the alpha- and beta-subunits of ATPsynthase on the endothelial cell surface is demonstrated using confocalmicroscopy with a monoclonal antibody specific for the alpha-subunit ofATP synthase and affinity-purified antibodies generated against therecombinant beta-subunit of ATP synthase (FIG. 9C). Theimmunofluorescence occurs in two distinctive patterns. First, there arenumerous fine punctate structures distributed over the entire cellsurface, except where the bulging nucleus displaces the plasma membranefrom the optical section. Second, each cell displays one or moreirregular clusters of punctate structures, suggesting an organizeddistribution on the cell surface. The cells were fixed in situ prior toaddition of antibodies to eliminate antibody-capping artifacts. Parallelstudies of immuno-localization between the alpha- and gamma-subunits ofATP synthase showed virtually identical patterns of co-localization(data not shown).

Fluorescence in all images was determined to be cell-surface associatedby three criteria: 1) permeabilized cells (FIG. 9D) produced adramatically different pattern characteristic of mitochondrial stainingof ATP synthase. Endothelial cell mitochondria are characteristicallytubular and reticular in pattern, with a peri-nuclear concentration asseen in Panel D. 2) Confocal optical sectioning along the z-axisconfirmed an apical concentration of antigen distribution characteristicof surface staining (FIGS. 10A-D, discussed below). 3) Co-staining witha known endothelial cell-surface marker, CD31, produced overlappingdistributions in z-axis optical sections, as would be expected forsurface localization (FIG. 10C). Flow cytometry of primary HUVEC wasalso performed with cells determined to be non-permeabilized by dyeexclusion. These cells demonstrated cell surface expression of thealpha-subunit of ATP synthase (data not shown) confirming that thestaining of ATP synthase that we detect is not due to permeabilizationbut rather to the presence of this enzyme on the cell surface.

The cell surface localization was further investigated by confocalmicroscopy comparing staining with CD31, an established endothelial cellsurface marker, and the alpha-subunit of ATP synthase. Additionalconfocal micrographs (not shown) were taken where the fluorescence fromthe red channel and from the green channel only were used. Thesemicrographs demonstrate CD31 and α-ATP synthase staining, respectively,while panel C shows the overlay of these two images. The image in panelC was taken at approximately the midpoint between the basal and apicalsurfaces of cultured endothelial cells and clearly shows a marginaldistribution of the punctate structures containing ATP synthase alongthe periphery of the cells. Panels A-D show the same field of view inoptical sections along the z-axis, with A starting near the basal aspectand D ending near the apical aspect of the cultured cells. It isimportant to note that the apical surface would be equivalent to thelumenal surface if these cells were within the vasculature. Althoughconfocal microscopy emphasizes structures within the optical plane ofsectioning, structures that are above or below the plane of section mayalso be visualized if their staining is particularly intense. However,structures that are outside the confocal plane will display fuzzymargins, while structures within the focal plane will show sharpmargins. This effect is seen with the α-ATP synthase staining.Examination of the sharp green spots demonstrates that essentially allα-ATP synthase staining is confined to the cellular margins in subapicalsections of non-permeabilized cells (Panels A-C). Moreover, A-ATPsynthase exhibits a greater intensity of staining in the apical section(Panel D), in which the confocal plane grazes the majority of theexposed apical surface, as would be expected for a surface-localizedmarker.

Angiostatin Binds to Bovine F₁ ATP Synthase. Human angiostatin bound topurified bovine F₁ ATP synthase passively adsorbed onto micro-titerwells in a concentration-dependent manner (FIG. 11). Human and bovineATP synthase are highly homologous, differing only by 8 amino acidresidues in the mature α-chains (SWISS-PROT accession numbers P25705 andP19483, respectively) and 6 residues in the mature β-chains (SWISS-PROTaccession numbers P06576 and P00829, respectively). Similar results wereobtained with purified bovine F₁ ATP synthase binding to immobilizedangiostatin (data not shown). Microtiter wells coated with decreasingconcentrations of purified bovine F₁ ATP synthase also showedconcentration-dependent binding of angiostatin (data not shown).Background binding of angiostatin to BSA-coated wells gave valuescomparable to the baseline seen in FIG. 11 (compare 0 μg/ml to 0.2μg/ml), indicating that angiostatin requires F₁ ATP synthase forefficient binding. Apparent dissociation constants (K_(d(app))) weredetermined from double reciprocal plots of the binding data as shown onthe inset of FIG. 11. The K_(d(app)) for angiostatin binding to purifiedF₁ ATP synthase is 12 nM. The holoenzyme associates with numerous otherproteins on the cell surface and thus may be sterically hindered to asignificant degree. In contrast, the purified F₁ subcomplex would not besubjected to steric hindrance by associated proteins and would beexpected to exhibit an increased affinity for its ligands.

Angiostatin Inhibits Purified Bovine F₁ ATP Synthase Activity. Althoughthe F₁-F_(o) ATP synthase holoenzyme efficiently catalyzes both theforward ATP synthase reaction and the reverse ATP hydrolysis reaction,the purified F₁ ATP synthase sub-complex only catalyzes the reversereaction. The ATP hydrolytic activity of the purified bovine F₁ ATPsynthase was measured using a coupled enzymatic assay in whichproduction of ADP is linked to oxidation of NADH via pyruvate kinase andlactate dehydrogenase, as showin in Zheng, J. & Ramirez, V. D. (1999)Eur J Pharmacol 368, 95-102. Thus, a decrease in the absorbance measuredat λ=340 nm indicates ATPase activity. Angiostatin completely inhibitedpurified ATPase activity, similar to a known F₁ inhibitor, NaN₃ (FIG.12). Polyclonal antibodies directed against the recombinant alpha-,beta-, and gamma-subunits of ATP synthase were also tested. Both alpha-and beta-subunit-specific ATP synthase polyclonal antibodies abolishedATPase activity. In contrast, the gamma-subunit-specific ATP synthaseantibody had no effect (data not shown). In addition, a commercialmonoclonal antibody specific for the alpha-subunit of ATP synthase(Molecular Probes) also inhibited activity. Pre-immune, unrelatedpolyclonal, and unrelated monoclonal antibodies employed as controls hadno effect on activity.

Surface-Associated ATP synthase is Catalytically Competent. The presenceof the alpha, beta and gamma-subunits of ATP synthase on the endothelialcell surface suggests that the entire catalytic complex is present. Totest for functional activity, we incubated HUVEC with [³H]ADP, [³²P_(i)]and unlabeled phosphate (100 mM), and followed ATP production byanalysis of supernatants on microcrystalline cellulose PEI TLC plates.ATP generation was detected within the first 15 s and reached maximallevels by 1 min. The possibility of ATP release from intracellular poolswas discounted because the cells tested were intact as evident by Trypanblue exclusion and lack of LDH release (data not shown). Moreover, theratio of [³H]ATP/[³²P]ATP remained constant at 1.56±0.04 over the timemeasured, thus demonstrating that the ADP and P_(i) substrates used toform ATP were derived exclusively from the external medium. No labeledproduct was detectable within the cell pellets (data not shown) againconfirming that the ATP was synthesized on the cell surface.

To determine whether angiostatin inhibits ATP synthesis on theendothelial cell surface, ATP production in the extracellular medium wasmeasured using a bioluminescence assay. This assay is highly specificfor ATP, to the exclusion of all other nucleotides. ATP concentrationssignificantly above basal levels were detected in the extracellularmilieu, indicating that de novo ATP synthesis occurred on the cellsurface (Table 3). Release of intracellular ATP pools was excluded bythe low level of ATP measured in the absence of ADP. Angiostatininhibited ATP synthesis in a dose-dependent manner (FIG. 13). ATPsynthesis was inhibited 81% by 1 μM angiostatin as shown in Table 3.TABLE 3 Inhibition of ATP Generation on the Surface of HUVEC as Measuredby Bioluminescent Luciferase Assay* Treatment applied to culturedPercent Inhibition HUVEC in the presence of 50 μM ADP (+/−S.E.M.) Mediumalone  0 Angiostatin (1 μM)   81 +/− 6/0 Polyclonal alpha-ATP synthase(1.0 mg/ml) 64.8 +/1 3.2 Polyclonal beta-ATP synthase (0.5 mg/ml) 56.8+/− 5.8 Pre-immune serum (1.0 mg/ml)   0 +/− 7.6 Oligomycin 83.5 +/− 4.5*Basal ATP levels in the assay medium (in the absence of ADP) producedbioluminescence signals equivalent to approximately 4% of theuninhibited levels in the presence of ADP.

Polyclonal antibodies against either the alpha or beta-subunits of ATPsynthase inhibited cell-surface production of ATP by 65% and 57%,respectively. In contrast, pre-immune serum showed no inhibition.Oligomycin, a known inhibitor of the F_(o) sub-complex, inhibited ATPsynthesis by 84% under these conditions, confirming that the majoractivity assayed was due to ATP synthase.

Inhibition of HUVEC Proliferation in the Presence of Angiostatin andanti- -ATP Synthase Antibodies. Polyclonal antibodies raised against thebeta-subunit of ATP synthase inhibited endothelial cell proliferation1.5-2-fold more effectively than angiostatin itself (Table 4). TABLE 4Inhibition of HUVEC Proliferation in the presence of Angiostatin andanti-betaATP Synthase antibody as Measured by CYQUANT Percent Inhibition(+/−S.E.M.) Medium alone   0 +/− 1.034 Angiostatin (1 μM) 57.2 +/− 1.16Cycloheximide (10 μg/ml)  100 +/− 7.0 Polyclonal beta-ATP synthase (100μg/ml) 80.9 +/− 15.9 Pre-immune serum (100 μg/ml)   0 +/− 1.7n = 3

The antiproliferative effect of the beta-subunit-specific antibodyapproached that of cycloheximide (80.9% and 100%, respectively).However, this effect is unlikely to represent a toxic response since thecellular morphology of the anti-beta-subunit ATP synthase treated cellswas unchanged from that of untreated cells, in contrast to the obviousrounded morphology caused by cycloheximide (data not shown). Thepreimmune serum exhibited no effect on proliferation. These data suggestthat antibodies directed against the beta-subunit of ATP synthase act asangiostatin mimetics in both biochemical and cell-based assays of ATPsynthase function.

Discussion

This study demonstrates that ATP synthase is catalytically active on theendothelial cell surface and that angiostatin-mediated inhibition ofthis activity correlates with inhibition of proliferation. Moreover,certain antibodies directed against the alpha and beta-subunits of ATPsynthase also inhibit enzymatic activity and endothelial cellproliferation. These antibodies, therefore, appear to constitutefunctional mimetics of angiostatin.

In addition to angiostatin, there are known inhibitors of ATP synthasethat exhibit anti-tumor effects including piceatannol and resveratrol.Resveratrol inhibits the development of DMBA-induced preneoplasticlesions and tumor growth (30). Piceatannol was shown to inhibit tumorcell growth by inhibition of protein-tyrosine kinases. The fact thatboth of these anti-tumor compounds inhibit ATP synthase suggests arelationship between the endothelial cell antiproliferative effects ofangiostatin and cell-surface associated ATP synthesis.

The generally accepted concept that ATP synthesis is strictly anintracellular process now appears questionable. It is well establishedthat both nucleosides and nucleotides act as extracellular signalingmolecules. Indeed, the extracellular role of nucleotides in regulatingmulticellular communication is widely conserved in eukaryotic evolution.Extracellular receptors for ATP exist as both ion channels (P2×) andG-protein coupled receptors (P2Y) that are ubiquitous to all mammaliantissues. A recent study demonstrates that ATP release by physical orchemical means contributes to the set point of cellular signalingpathways coupled to P2Y receptors (Ostrom, R. S., Gregorian, C. & Insel,P. A. (2000) J Biol Chem 275, 11735-9). In the cardiovascular system,adenosine released from myocytes maintains blood flow to ischemic areasof the heart, and ATP may play a role in the moment-to-moment regulationof cardiac blood flow in non-pathological states. Endothelial cellsrelease ATP in response to stimuli such as shear stress and vasoactiveagonists including ATP itself.

The immunolocalization data show that the alpha, beta and gamma-subunitsof ATP synthase are present on the endothelial cell surface where theyextensively co-localize. These subunits represent the minimal componentsrequired for efficient ATPase catalytic activity and strongly suggestthat the cell-surface form of ATP synthase is similar to themitochondrial form. Moreover, cell-surface ATP synthase is present indiscrete foci, indicating structural organization of the enzyme complex.Numerous other mitochondrial matrix enzymes that co-localize with ATPsynthase into these discrete foci on the endothelial cell surface werealso detected, but mitochondrial outer membrane markers were notdetected (data not shown). These findings suggest that a highly complexenergy production apparatus exists on the endothelial cell surface.

The data strongly suggest that angiostatin operates through inhibitionof the enzymatic activity of cell-surface ATP synthase. Data supportingthis interpretation include concordance of inhibitory activities ofangiostatin, known small molecule inhibitors of ATP synthase, andantibodies specific for ATP synthase subunits in binding and biochemicalassays using purified bovine enzyme. There are also similar effects ofangiostatin and subunit-specific antibodies in inhibition of ATPproduction on the surface of cultured human endothelial cells. It shouldbe noted that existing small molecule inhibitors cannot be used in thisassay because they also inhibit the mitochondrial form of ATP synthase,whereas neither angiostatin nor the subunit-specific antibodies crossthe cell membrane. Finally, angiostatin and anti-beta-subunit antibodiesshow similar ability to inhibit proliferation of cultured humanendothelial cells.

Collectively, these data suggest a steric hindrance model of ATPsynthase inhibition by angiostatin. ATP synthase couples proton fluxacross a membrane to rotation of the gamma-subunit, which in turninduces cyclical conformational changes in the catalytic beta-subunitBoyer, P. D. (1997) Annu Rev Biochem 66, 717-49. These conformationalchanges, required for conversion of ADP to ATP, can be blocked by anycompound that either inhibits rotation of the gamma-subunit or locks theconformation of the alpha or beta-subunits. The binding of specificantibodies can lock the conformation of flexible antigens. Thus, bothangiostatin and certain antibodies to the alpha or beta-subunits of ATPsynthase inhibit the enzyme by blocking conformational changes of theenzyme complex required for ATP synthesis or hydrolysis. Functionalmimetics of angiostatin can be derived by developing monoclonalantibodies against the alpha or beta-subunits of ATP synthase. Suchantibodies will be far easier to produce and administer than angiostatinand offer significant advances in anti-angiogenic therapy of cancer andother proliferative diseases.

All documents cited above are hereby incorporated in their entirety byreference. From the foregoing, it will be obvious to those skilled inthe art that various modifications in the above-described methods, andcompositions can be made without departing from the spirit and scope ofthe invention. Accordingly, the invention may be embodied in otherspecific forms without departing from the spirit or essentialcharacteristics thereof. Present embodiments and examples, therefore,are to be considered in all respects as illustrative and notrestrictive, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

1. A composition for use in anti-angiogenic or angiogenic therapycomprising: a) an angiostatin agonist, partial agonist, inverse agonist,antagonist and/or angiostatin allosteric modulator, and b) a suitablecarrier.
 2. The composition of claim 1, wherein the angiostatin agonist,partial agonist, inverse agonist, antagonist and/or angiostatinallosteric modulator are selected from the group consisting ofantibodies, antibody fragments, enzymes, peptides and oligonucleotides.3. The composition of claim 1, wherein the angiostatin agonist,angiostatin partial agonist, inverse agonist and/or angiostatinallosteric modulator is a conjugate of an anti-tumor agent that does notbind to the alpha or beta subunits of F1 ATP synthase and a compoundthat does bind to the alpha or beta subunits of F1 ATP synthase.
 4. Thecomposition of claim 1, wherein the angiostatin agonist, partialagonist, inverse agonist, antagonist and/or angiostatin allostericmodulator is an antibody or an antibody fragment.
 5. The composition ofclaim 4, wherein the antibody is a monoclonal antibody or antibodyfragment thereof.
 6. The composition of claim 4, wherein the antibody isa humanized antibody or antibody fragment thereof.
 7. The composition ofclaim 1, wherein the angiostatin agonist, partial agonist, inverseagonist, antagonist and/or angiostatin allosteric modulator are presentin or conjugated onto a liposome or microparticle that is of a suitablesize for intraveneous administration but that lodges in capillary beds.8. The composition of claim 1, further comprising an anti-tumor agentthat does not bind to the alpha or beta subunits of FI ATP synthase. 9.The composition of claim 1, further comprising a COX-2 inhibitor. 10.The composition of claim 1, further comprising an angiogenesis-promotingagent that does not bind to the alpha or beta subunits of F1 ATPsynthase.
 11. A method of inhibiting angiogenesis, comprisingadministering to a patient in need of treatment thereof an effective,angiogenesis inhibiting amount of an angiostatin agonist, angiostatinpartial agonist or angiostatin allosteric promoter.
 12. The method ofclaim 11, wherein the angiostatin agonist, angiostatin partial agonistor angiostatin allosteric promoter is a compound selected from the groupconsisting of antibodies, antibody fragments, enzymes, peptides andoligonucleotides.
 13. The method of claim 11, wherein the angiostatinagonist, angiostatin partial agonist or angiostatin allosteric promoteris a conjugate of an anti-tumor agent that does not bind to the alpha orbeta subunits of F1 ATP synthase and an angiostatin agonist, angiostatinpartial agonist or angiostatin allosteric promoter.
 14. The method ofclaim 11, wherein the angiostatin agonist, angiostatin partial agonistor angiostatin allosteric promoter is an antibody or an antibodyfragment.
 15. The method of claim 14, wherein the antibody is amonoclonal antibody or antibody fragment thereof.
 16. The method ofclaim 14, wherein the antibody is a humanized antibody or antibodyfragment thereof.
 17. The method of claim 11, wherein the angiostatinagonist, angiostatin partial agonist or angiostatin allosteric promoterare present in or conjugated onto a liposome or microparticle that is ofa suitable size for intraveneous administration but that lodges incapillary beds.
 18. The method of claim 11, further comprisingadministering an anti-tumor agent that does not bind to the alpha orbeta subunits of F1 ATP synthase.
 19. The method of claim 11, furthercomprising administering a COX-2 inhibitor.
 20. The method of claim 11,wherein the angiostatin agonist, angiostatin partial agonist orangiostatin allosteric promoter is administered intravenously,intramuscularly, intradermally or subcutaneously.
 21. A method ofpromoting angiogenesis, comprising administering to a patient in need oftreatment thereof an effective, angiogenesis-promoting amount of anangiostatin antagonist or an angiostatin allosteric inhibitor.
 22. Themethod of claim 21, wherein the angiostatin antagonist or an angiostatinallosteric inhibitor is a compound selected from the group consisting ofantibodies, antibody fragments, enzymes, peptides, and oligonucleotides.23. The method of claim 21, wherein the angiostatin antagonist orangiostatin allosteric inhibitor is a conjugate of anangiogenesis-promoting compound that does not bind to the alpha or betasubunits of F1 ATP synthase and an angiostatin antagonist or angiostatinallosteric inhibitor.
 24. The method of claim 21, wherein theangiostatin antagonist or angiostatin allosteric inhibitor is anantibody or antibody fragment.
 25. The method of claim 24, wherein theantibody is a monoclonal antibody or antibody fragment thereof.
 26. Themethod of claim 24, wherein the antibody is a humanized antibody orantibody fragment thereof.
 27. The method of claim 21, wherein theangiostatin antagonist or angiostatin allosteric inhibitor are presentin or conjugated to a liposome or microparticle that is of a suitablesize for intraveneous administration but that lodges in capillary beds.28. The method of claim 21, further comprising administering anangiogenesis-promoting agent that does not bind to the alpha or betasubunits of F1 ATP synthase.
 29. The method of claim 21, wherein theangiostatin antagonist or angiostatin allosteric inhibitor isadministered intravenously or intramuscularly.
 30. The method of claim21, wherein the angiostatin antagonist or angiostatin allostericinhibitor is administered locally to a location in a patient in need ofincreased vascularization.
 31. A method of screening a test compound forits ability to inhibit or enhance the binding of angiostatin to ATPsynthase comprising: i) contacting the test compound and angiostatinwith the alpha and/or beta subunits of ATP synthase under conditionssuch that angiostatin can bind to the subunits in the absence of thetest compound, and ii) determining the amount of angiostatin bound tothe subunits, and comparing that amount to an amount of angiostatinbound to the subunits in the absence of the test compound, wherein areduction in the amount of angiostatin bound to the alpha and/or betasubunits of ATP synthase in the presence of the test compound indicatesthat the test compound inhibits the binding of angiostatin to thesubunits, and wherein an increase of the amount of angiostatin bound tothe alpha and/or beta subunits of ATP synthase in the presence of thetest compound indicates that the test compound enhances the binding ofangiostatin to the subunits.
 32. The method of claim 31 wherein theangiostatin bears a detectable label.
 33. The method of claim 31 whereinthe alpha and/or beta subunits of ATP synthase are attached to a solidsupport.
 34. The method of claim 31 wherein the alpha and/or betasubunits of ATP synthase are associated with a lipid membrane.
 35. Themethod of claim 34 wherein the membrane is a membrane of an intact cell.36. The method of claim 35 wherein the cell naturally expresses ATPsynthase.
 37. The method of claim 35 wherein the cell has beentransformed with one or more nucleic acid sequence that encode thealpha, beta, delta, gamma and/or epsilon subunits of ATP synthase.
 38. Acompound identified in the method of claim 31 as inhibiting the bindingof angiostatin to the alpha and/or beta subunits of ATP synthase.
 39. Acompound identified in the method of claim 31 as enhancing the bindingof angiostatin to the alpha and/or beta subunits of ATP synthase.
 40. Amethod of screening a test compound for its ability to modulate abioactivity resulting from binding of angiostatin to the alpha and/orbeta subunits of ATP synthase comprising: i) contacting the testcompound and angiostatin with a cell that expresses the alpha and/orbeta subunits of ATP synthase under conditions such that angiostatin canbind to the subunits in the absence of the test compound, and ii)determining the amount of angiostatin required to achieve the samebioactivity in the presence of the test compound as in the absence ofthe test compound, wherein a reduction in the amount of angiostatinrequired to achieve the same bioactivity in the presence of the testcompound indicates that the test compound is an angiostatin agonist,partial agonist or allosteric inhibitor, and wherein an increase in theamount of angiostatin required to achieve the same bioactivity in thepresence of the test compound indicates that the test compound is anangiostatin antagonist or allosteric promoter.
 41. An angiostatinagonist, partial agonist or allosteric inhibitor identified inaccordance with the method of claim
 40. 42. An angiostatin antagonist orallosteric promoter identified in accordance with the method of claim40.
 43. The method of claim 40 wherein the bioactivity is inhibition ofendothelial cell proliferation, migration, tube formation, cell surfaceATP synthesis, ATP hydrolysis, in vivo inhibition of angiogenesis, or invivo inhibition of tumor growth.
 44. The method of claim 40 wherein thebioactivity is enhancement of proton pumping.
 45. An expressionconstruct comprising a vector and a nucleic acid sequence encoding thealpha and/or beta subunits of ATP synthase, operably linked to apromoter.
 46. A host cell comprising the construct of claim
 45. 47. Amethod of producing the alpha and/or beta subunits of ATP synthase,comprising culturing the host cell of claim 46 under conditions suchthat the nucleic acid is expressed and the alpha and/or beta subunits ofATP synthase are thereby produced.
 48. A monoclonal antibody or antibodyfragment thereof specific for the alpha and/or beta subunits of ATPsynthase that functions as an angiostatin agonist.
 49. A monoclonalantibody or antibody fragment thereof specific for the alpha and/or betasubunits of ATP synthase that functions as an angiostatin partialagonist.
 50. A monoclonal antibody or antibody fragment thereof specificfor the alpha and/or beta subunits of ATP synthase that functions as anangiostatin inverse agonist.
 51. A monoclonal antibody or antibodyfragment thereof specific for the alpha and/or beta subunits of ATPsynthase that functions as an angiostatin antagonist.
 52. A monoclonalantibody or antibody fragment thereof specific for the alpha and/or betasubunits of ATP synthase that functions as an angiostatin allostericmodulator.